Detection of DNA that originates from a specific cell-type and related methods

ABSTRACT

The present invention relates to methods to detect an amount of DNA that originates from cells of a given type, where the sample comprising such DNA in admixture with DNA that does not originate from such cells. Such methods are based on differential methylation, at certain regions, of the DNA that originates from the given type of cells compared to the admixed DNA. Such methods have particular application in the detection, from a biological fluid from a pregnant female, of cell free DNA that originates from a foetus or the placenta of a foetus, or the detection, from a biological fluid from an individual, of cell free DNA that originates from cells of a tumour. Accordingly, such methods have diagnostic, prognostic and/or predictive utility for detecting an increased risk of an individual suffering from or developing a medical condition such as preeclampsia or cancer, and/or to aid (subsequent) diagnostic, prognostic and/or predictive methods such as the detection of chromosomal trisomy in a foetus, including for twin-pregnancies. The present invention also relates to compositions, kits, computer program products and other aspects that are used in, useful for or related to the practice of such methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage application ofPCT/EP2015/060188 filed 8 May 2015, which claims priority to EuropeanApplication No. 14167769.0 filed 9 May 2014 and European Application No.14167775.7 filed 9 May 2014, the entire disclosures of which are herebyincorporated by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 17, 2017, isnamed DFMP-115-US_SL.TXT and is 1,392,495 bytes in size.

The present invention relates to methods to detect an amount of DNA thatoriginates from cells of a given type, where the sample comprising suchDNA in admixture with DNA that does not originate from such cells. Suchmethods are based on differential methylation, at certain regions, ofthe DNA that originates from the given type of cells compared to theadmixed DNA. Such methods have particular application in the detection,from a biological fluid from a pregnant female, of cell free DNA thatoriginates from a foetus or the placenta of a foetus, or the detection,from a biological fluid from an individual, of cell free DNA thatoriginates from cells of a tumour. Accordingly, such methods havediagnostic, prognostic and/or predictive utility for detecting anincreased risk of an individual suffering from or developing a medicalcondition such as preeclampsia or cancer, and/or to aid (subsequent)diagnostic, prognostic and/or predictive methods such as the detectionof chromosomal trisomy in a foetus, including for twin-pregnancies. Thepresent invention also relates to compositions, kits, computer programproducts and other aspects that are used in, useful for or related tothe practice of such methods.

Cell-free DNA (cfDNA), especially that found in plasma or serum, hasbeen the subject of considerable research over the past decade. Despitethe original finding of circulating cell-free nucleic acids in thebloodstream being described by Mandel and Metais as early as 1948(Mandel and Metais 1948, CR Acad Sci Paris 142:241), it took until themid 1990s for proof that tumours shed DNA into the circulatory system(Sorenson et al 1994, Cancer Epidemiol Biomarkers Prev 3:67; Vassioukhinet al 1994, Br J Haematol 86:774), and until 1997 for the discovery ofcfDNA originating from a foetus in the circulatory system of the mother(Lo et al 1997, Lancet 350:485).

Among other forms of characteristics shown by circulating cfDNA,numerous studies have described the presence of methylated circulatingcfDNA in the plasma/serum and other body fluids of patients with varioustypes of malignancy and the absence of methylated DNA in normal controlpatients (for review see Muller and Widschwendter 2003, Expert Rev MolDiagn 3:443). Although other characteristics of circulating cfDNA existand are important for diagnostic, prognostic or predictive studies (forexample, sequence mutations and micro duplications/deletions), suchmethylation-based epigenetic characteristics have become an increasinglyimportant source of serologic markers for diagnosis, risk assessment andeven for therapy monitoring during follow-up of cancer patients.

Likewise, the use of differences in foetal cfDNA present in the maternalcirculation has been the main goal for the development of non-invasiveprenatal tests (NIPT). Foetal cfDNA is derived from embryonic celldegradation in maternal peripheral blood (Lo et al 2000, Clin Chem46:1301) or from apoptotic placental cells (Smid et al 2006, PrenatDiagn 26:785). It has been demonstrated that foetal cfDNA from maternalplasma is cleared immediately (within a few hours) after pregnancy (Loet al 1999, Am J Hum Genet 64:218). This finding is of great importance,since the presence of foetal cfDNA from previous pregnancies wouldotherwise interfere with the correct interpretation of subsequentpregnancies.

It is believed that 60% of tissue-specific differentially methylatedregions are methylated in embryonic cells, while during thedifferentiation of embryonic tissues to adult tissues, they undergode-methylation (Kawai et al 1993, Nucleic Acids Res 21:5604). Based onthe evidence that foetal cfDNA in maternal plasma is of placentalorigin, epigenetic differences between maternal peripheral (whole) bloodand placental DNA have been used to detect a hypomethylated genesequence (maspin/SERPINB5) in maternal plasma derived from the foetus(Masuzaki et al 2004, J Med Genet 41:289; Fiori et al 2004, Hum Reprod19:723; Chim et al 2005, Proc Natl Acad Sci USA 102:14753).Subsequently, a number of additional differential foetalmethylation-based epigenetic molecular markers have been described,including the RASSF1A gene on chromosome 3, as well as a marker onchromosome 21 (Chiu et al 2007, Am J Pathol 170:941; Old et al 2007,Reprod Biomed Online 15:22; Chim et al 2008, Clin Chem 54:500) andothers including T-box 3 (TBX3) (Nygren et al 2010, Clin Chem 65:10; WO2010/033639; WO 2011/034631).

Various methodologies exist for NIPT based on the analysis of foetalcfDNA. For example, foetal sex determination using eg DYS14 (Lo et al1997; Lancet 350:485), as well as foetal Rhesus D found in maternalcirculation in pregnancies in which the mother was Rhesus D negative (Lo1998, N Eng J Med 339:1734). Also, and of particular relevance, arethose using next generation sequencing (NGS) technologies on cfDNAisolated from maternal plasma with the primary aim of detecting the mostcommon chromosomal aneuploidies as commercially available tests (forexample, those using random massively parallel sequencing:www.sequenom.com; www.lifecodexx.com; www.verinata.com). Othertechnologies include targeted approaches, the aim of which is to enrichspecific genomic regions of interest before sequencing to reduce thenumber of sequence tags needed to perform a reliable statisticalanalysis (eg www.ariosadx.com or www.natera.com), polymorphism analysisor digital PCR (for review, see Go et al 2011, Human Reprod Update17:372). However, regardless of the specific technology used, currentapplications of NIPT rely on the qualitative detection of foetal cfDNAto determine the genetic makeup of the foetus. Such an approach leads toan analytic dilemma, because test results from samples that do notcontain any or sufficient foetal DNA or are contaminated with maternalcellular DNA can be misleading. The analogous issue arises indiagnostic, prognostic or predicative tests of tumour derived cfDNA fromthe circulatory system: the quality of the test result often isdependent on the presence of sufficient, or sufficiently pure,tumour-derived cfDNA in the total DNA from the sample.

The quantitative determination of an amount of DNA originating from sucha cell type may, in itself, form a critical part of a diagnostic,prognostic or predicative test. For example, even though studies havedemonstrated that the amount of foetal DNA released in maternalcirculation increases with pregnancy progression (Lo et al 1998, Am JHum Genet 62:768), preeclampsia, which results from abnormal trophoblastinvasion, is also associated with further elevated foetal cfDNA levelsin the maternal circulation. Lo et al (1999, Clin Chem 45:184)demonstrated a fivefold increase in circulating foetal cfDNAconcentrations in the plasma of symptomatic preeclamptic women comparedwith control pregnant subjects, and further studies have investigated ifelevated serum foetal cfDNA developed into early-onset preeclampsia (Yuet al 2013, Int J Mol Sci 14:7571), and the potential of cfDNA as amarker for preeclampsia is being increasingly studied (for review, seeHahn et al 2011, Placenta 32(Supl):S17). An increased level ofcirculating cfDNA and/or the level of methylation of such DNA at certainregions is also associated with other medical conditions. For example,hypermethylation of serum cfDNA was found to be common in patientssuffering from oesophageal squamous cell carcinoma, and diagnosticaccuracy was increased when methylation of multiple genes (RAR-beta,DAPK, CDH1, p16 and RASSF1A) were analysed in combination (Li et al2011, Epigenetics 6:307). Elevated levels of circulating cfDNA have beenreported in patients with acute dengue virus infection (Ha et al 2011,PLoS One 6(10):e25969), in acute Puumala hantavirus infection Outinen etal 2012, PLoS One 7(2):e31455) and high cfDNA has been reported topredict fatal outcome among Staphylococcus aureus bacteraemia patientswith intensive care unit treatment (Forsblom et al 2014, PLoS One 10;9(2):e87741).

It is known that foetal cfDNA present in the maternal circulatory systemand tumour derived circulating cfDNA is degraded. For example, studiescharacterising cfDNA in maternal plasma have found that the size offoetal DNA fragments were estimated to be <0.3 kb, whereas that ofmaternal DNA was >1 kb (Chan et al 2004, Clin Chem 50:88). Follow-upstudies have demonstrated that the release of foetal DNA is due to theapoptosis of no more than three nucleosomal complexes, it has also beenshown that the average foetal fragment size is 286+/−28 bp with amaximum foetal cfDNA fragment size ranging from 219 to 313 bp (Kimura etal 2011, Nagoya J Med Sci 73:129), and another study has reported thatthe most significant difference in the size distribution between foetaland total DNA is that foetal DNA exhibits a reduction in a 166-bp peaksize and a relative prominence of the 143-bp peak; the latter likelycorresponding to the trimming of a ˜20-bp linker fragment from anucleosome to its core particle of ˜146 bp (Lo et al 2010, Sci TranslMed 2:61).

In cancer patients, circulating cfDNA in plasma is protein-bound(nucleosomal) DNA and has a short half-life (10 to 15 min) which isremoved mainly by the liver (Elshimali et al 2013, Int J Mol Sci14:18925). Accumulation of cfDNA in the circulation of cancer patientscan result from an excessive release of DNA caused by massive celldeath, inefficient removal of the dead cells, or a combination of both(Zeerleder 2006, Crit Care 10:142). It should be noted that althoughcancer patients requiring renal support have higher values ofcirculating cfDNA, the renal elimination is not the main mechanism ofits clearance. The plasma levels of circulating cfDNA do not seem to bedramatically altered in chronic kidney disease, peritoneal dialysis orhemodialysis (Kirsch et al 2008, Ann NY Acad Sci 1137:135).

Although the nucleosome is a very stable protein-DNA complex, it is notstatic and has been shown to undergo a number of different structuralre-arrangements including nucleosome sliding and DNA site exposure.Depending on the context, nucleosomes can inhibit or facilitatetranscription factor binding. Also, packaging of DNA into nucleosomesvaries depending on the cell cycle stage and by local DNA region(Russell 2010, ‘iGenetics”. 3rd ed. San Francisco: Pearson BenjaminCummings, pp 24-27). The degree to which chromatin is condensed isassociated with a certain transcriptional state. Unpackaged or loosechromatin is more transcriptionally active than tightly packagedchromatin because it is more accessible to transcriptional machinery. Byremodelling chromatin structure and changing the density of DNApackaging, gene expression can thus be modulated. Accordingly, andwithout being bound by theory, the qualitative and/or quantitative levelof chromatin packing of a given region of cfDNA may affect itsstability, and hence the amount detected in the circulatory system atany given time, Correspondingly, differences between the level ofchromatin packing between different DNA regions (for example, due todifferences in each regions state of transcription) may influence therelative quantities of DNA from each of these regions when detected ascfDNA, particularly as two studies have investigated in more detail thekinetics of, and reported the rapid, clearance of cfDNA from thecirculatory system (Gauthier et al 1996, J Immunol 156:1151; Lo et al1999, Am J Hum Genet 64:218).

Various prior art methods have been described to detect, and quantify,cfDNA from a specific cell type. Quantitative analysis of aberrant p16methylation was described using probe-based real-time quantitative PCR(Lo et al 1999, Cancer res 59:3899). Analogously, differences in themethylation of the placental maspin gene found in material plasma hasbeen described, and the methylation signature further analysed usingMALDI-TOF mass-spectrometry (Chim et al 2005). Total cfDNA and that frommale foetuses (only) were accurately and robustly quantified in materalplasma from 5 to 41 weeks of gestation using a Y-chromosome specificmarker (SRY) (Birch et al 2005, Clin Chem 51:2). Hypermethylation ofRASSF1A has been proposed as a universal foetal DNA marker to improvethe reliability of NIPT, and was studied in a duplex probe-basedreal-time PCR reaction compared to the non-differentially methylatedregion on the beta-actin gene (Chan et al 2006, Clin Chem 52:12). Acomplex method of quantification has been described (Nygren et al 2010;Clin Chem 56:10; WO 2010/033639; WO 2011/034631): starting from a13-plex competition-PCR reaction (5 differentially methylated regions(DMRs) including TBX3, 3 regions on different genes for total DNAquantification, 3 for quantification of chromosome Y and 2 forrestriction enzyme controls), such a complex reaction is subsequentlyprocessed for singe-base extension reactions and finallymass-spectrometry is subsequently conducted to both quantify andidentify each of the single alleles my mass differences. Also using acomplex process starting from methylated DNA immunoprecipitation, andbased on SYBR green based quantitative PCR of a plurality of DMRs, hasbeen claimed to be able to accurately quantitate foetal cfDNA and usesuch quantitation from eg chromosome 21 DMRs, to prenatally diagnosetrisomies (Papageorgiou et al 2011, Nat Med 4:510; WO 2012/092592);although technical concerns about such an approach to diagnose trisomieshave been raised (Tong et al 2012; Nat Med 18:1327). High-throughputdroplet digital PCR (ddPCR) has been described for absolutequantification of DNA copy number from normal and tumorous breasttissues, and also total and foetal cfDNA in maternal plasma using duplexprobe-based quantitative PCR of RASSF1/RNaseP and RASSF1/beta-actin(Hindson et al 2011, Anal Chem 83:8604). Separate SYBR greenquantitative PCR reactions of RASSF1A, SRY and DYS14 have been evaluatedas an assay to detect RASSf1A to facilitate improved diagnosticreliability of NIPT (White et al 2012; PLOS ONE 7(9):e45073). However,generally considered as the “gold standard” for the quantitativemeasurement of foetal cfDNA against which other assays are oftencompared, remains the quantification of Y chromosome-specific genes (egSFY) of male foetuses eg, as used by Yu and co-workers to determinewhether the increased foetal cfDNA in maternal serum level of gravitasdeveloped into early-onset preeclampsia (Yu et al 2013, Int J Mol Sci14:7571).

Others have suggested that epigenetic biomarkers can be exploited forNIPT of trisomy 21, such as by quantifying a chromosome 21-derivedsequence in epigenetically identified foetal cfDNA, relative to areference sequence derived from another autosome or sex chromosome (Oldet al 2007; RBMOnline 15:227); where the comparative reference sequencemay be either a SNP allele-specific region or by direct comparison witha foetal- or placental-specific methylation marker on the referencechromosome (Tong et al 2006; ClinChem 52:2194; WO2007/132166;WO2007/132167; Chim et al 2008; ClinChem 54:500; Papageorgio et al 2009;AmJPath 174:1609).

Accordingly there is a need, from one or more of the above orperspectives, for improved methods to detect, preferably quantitatively,an amount of a species of DNA that originates from a particular celltype, such as a tumour-, foetal- or a placental cell, in particular toso detect cfDNA eg from the circulatory system of an individual. Inparticular, there is also a need, from one or more of the above orperspectives, for improved methods to detect, indicate or diagnose thepresence of an abnormality in such a species of DNA, for example achromosomal abnormality such as a chromosomal aneuploidy in a foetus.

Accordingly, it is an object of the present invention to providealternative, improved, simpler, cheaper and/or integrated means ormethods that address one or more of these or other problems. Such anobject underlying the present invention is solved by the subject matteras disclosed or defined anywhere herein, for example by the subjectmatter of the attached claims.

Generally, and by way of brief description, the main aspects of thepresent invention can be described as follows:

In a first aspect, and as may be further described, defined, claimed orotherwise disclosed herein, the invention relates to a method fordetecting in a sample from an individual an amount of a species of DNAoriginating from cells of a given type, which sample comprises saidspecies of DNA in admixture with differently methylated DNA notoriginating from cells of said type; said method comprising the steps:

-   (a) treating the DNA present in said sample with a reagent that    differentially modifies methylated and non-methylated DNA;-   (b) detecting in said sample the presence of methylation in said    species of DNA at one or more differentially methylated region(s)    (DMR(s)) that is(are) differently methylated between said species of    DNA and the DNA not originating from cells of said type, the    modification of DNA of such DMR by said reagent is sensitive to    methylation of DNA, wherein the presence of methylated DNA at said    DMR indicates the presence of said amount of species of DNA in said    sample and the absence of methylated DNA at said DMR indicates the    absence of said species of DNA in said sample; and-   (c) detecting an amount of total DNA present in said sample using at    least one other region that is not differently methylated between    said species of DNA and the DNA not originating from cells of said    type, the modification of which regions(s) by said reagent is    insensitive to methylation of DNA, wherein, said other region is    located between about 20 bp and about 20 kb upstream or downstream    of said DMR.

In a second aspect, and as may be further described, defined, claimed orotherwise disclosed herein, the invention relates to a method fordetecting in a sample from an individual an amount of a species of DNAoriginating from cells of a given type, which sample comprises saidspecies of DNA in admixture with differently methylated DNA notoriginating from cells of said type; said method comprising the steps

-   (a) treating the DNA present in said sample with a reagent that    differentially modifies methylated and non-methylated DNA;-   (b) detecting in said sample the presence of methylation in said    species of DNA at two or more differentially methylated regions    (DMRs) that are differently methylated between said species of DNA    and the DNA not originating from cells of said type, the    modification of DNA of such DMRs by said reagent is sensitive to    methylation of DNA, wherein the presence of methylated DNA at one or    more of said DMRs indicates the presence of said amount of species    of DNA in said sample and the absence of methylated DNA at said DMRs    indicates the absence of said species of DNA in said sample; and-   (c) detecting an amount of total DNA present in said sample using at    least one other region that is not differently methylated between    said species of DNA and the DNA not originating from cells of said    type, the modification of which region(s) by said reagent is    insensitive to methylation of DNA,    wherein, said detection in step (b) and said detection in step (c)    are made using the same aliquot of DNA of said sample, and in the    same vessel, and effectively simultaneously for such DMRs and other    region(s), and using: (x) the same detectable label(s) for each of    said DMRs; and (y) a different detectable label(s) for said other    region(s).

In a further aspect, and as may be further described, defined, claimedor otherwise disclosed herein, the invention relates to a method fordetecting a chromosomal aneuploidy in a foetus carried by a pregnantfemale, said method comprising the steps:

-   (A) Determining, using a method of the first or second aspect of the    present invention, in a sample taken from said pregnant female the    amount of a first species of DNA that originates from cells of a    foetus and/or the placenta of a foetus, wherein said first species    of DNA is located on a chromosome relevant to the chromosomal    aneuploidy or within a section of a chromosome relevant to the    chromosomal aneuploidy, and wherein said first species of DNA that    originates from cells of a foetus and/or the placenta of a foetus is    distinguished from its counterpart of maternal origin in the sample    due to differential DNA methylation;-   (B) Determining, using a method of the first or second aspect of the    present invention, the amount of a second species of DNA that    originates from cells of a foetus and/or the placenta of a foetus in    said sample, wherein said second species of DNA is located on a    reference chromosome, and wherein said second species of DNA that    originates from cells of a foetus and/or the placenta of a foetus is    distinguished from its counterpart of maternal origin in the sample    due to differential DNA methylation;-   (C) determining the relative amount, preferable the ratio, of the    amounts from (A) and (B); and-   (D) comparing said relative amount or ratio with a threshold and/or    reference distribution of amount(s) or ratio(s), wherein: a relative    amount or ratio that is higher or lower than said threshold and/or    reference distribution of amount(s) or ratio(s) indicates the    presence of the chromosomal aneuploidy in the foetus.

In another aspect, the invention also relates to a method for detectingan increased risk of an individual suffering from or developing amedical condition, said method comprising the steps:

-   (i) conducting a method of the first or second aspect of the    invention, wherein each of the detection steps comprises    quantitative detection; and-   (ii) comparing the amount of said species of DNA detected with a    threshold amount and/or a reference distribution of amounts,

wherein an increase in, or outlying of, the amount of said species ofDNA indicates an increased risk of the individual suffering from ordeveloping said medical condition. In other aspects, the invention alsorelates to a composition, a kit and a computer program product, in eachcase as may be described, defined, claimed or otherwise disclosedherein, for use within or in connection with a method of the invention.

The figures show:

FIG. 1 depicts: (a) a schematic representation of the differentiallymethylated region(s) (“DMR”) and other regions(s) (“OR”) used in themethod of the first aspect of the invention; and (b) a schematicrepresentation of the differentially methylated regions (“DMR”) andother regions(s) (“OR”) used in the method of the second aspect of theinvention.

FIG. 2 depicts a schematic representation of the differentiallymethylated regions (“DMR”) and other regions (“OR”) used in Example 1.

FIG. 3 depicts the correlation of the amount of male specific DNA (Ychromosomal-representation) to the foetal cfDNA fraction measured by amethod of the present invention (Example 1) for study twin cases withknown foetal genders.

FIG. 4 depicts the improved sensitivity of a method of the inventioncompared to foetal cfDNA fraction detected using separate reactions of asingle DMR. The number of PCR cycles (Cp) required for detection offoetal cfDNA (Example 2) in a sample using either RASSF1A or TBX3 aloneas a single DMR, or as a multiplex (using the same labels) of RASSF1Aand TBX3.

FIG. 5 depicts a schematic representation of the operations conducted bya computer program product of the invention.

FIG. 6 depicts a schematic representation of the differentiallymethylated regions (“DMR”) and other regions (“OR”) used in EXAMPLE 5,and based on a method of the first aspect of the present invention.

FIG. 7 depicts a schematic representation of the differentiallymethylated regions (“DMR”) and other regions (“OR”) based on a method ofthe second aspect of the present invention.

FIG. 8 depicts the results of a z-score analysis of the ratio of foetalchromosome 21 to foetal chromosome 12 of 138 cfDNA samples taken frompregnant females which include 8 such samples from pregnancies carryinga foetus with a chromosome 21 trisomy.

The present invention, and particular non-limiting aspects and/orembodiments thereof, can be described in more detail as follows:

In a first aspect, the invention relates to a method for detecting in asample from an individual an amount of a species of DNA originating fromcells of a given type, which sample comprises said species of DNA inadmixture with differently methylated DNA not originating from cells ofsaid type; said method comprising the steps:

-   (a) treating the DNA present in said sample with a reagent that    differentially modifies methylated and non-methylated DNA;-   (b) detecting in said sample the presence of methylation in said    species of DNA at one or more differentially methylated region(s)    (DMR(s)) that is(are) differently methylated between said species of    DNA and the DNA not originating from cells of said type, the    modification of DNA of such DMR by said reagent is sensitive to    methylation of DNA, wherein the presence of methylated DNA at said    DMR indicates the presence of said amount of species of DNA in said    sample and the absence of methylated DNA at said DMR indicates the    absence of said species of DNA in said sample; and-   (c) detecting an amount of total DNA present in said sample using at    least one other region that is not differently methylated between    said species of DNA and the DNA not originating from cells of said    type, the modification of which regions(s) by said reagent is    insensitive to methylation of DNA,    wherein, said other region is located between about 20 bp and about    20 kb upstream or downstream of said DMR.

In a second aspect, the invention relates to a method for detecting in asample from an individual an amount of a species of DNA originating fromcells of a given type, which sample comprises said species of DNA inadmixture with differently methylated DNA not originating from cells ofsaid type; said method comprising the steps:

-   (a) treating the DNA present in said sample with a reagent that    differentially modifies methylated and non-methylated DNA;-   (b) detecting in said sample the presence of methylation in said    species of DNA at two or more differentially methylated regions    (DMRs) that are differently methylated between said species of DNA    and the DNA not originating from cells of said type, the    modification of DNA of such DMRs by said reagent is sensitive to    methylation of DNA, wherein the presence of methylated DNA at one or    more of said DMRs indicates the presence of said amount of species    of DNA in said sample and the absence of methylated DNA at said DMRs    indicates the absence of said species of DNA in said sample; and-   (c) detecting an amount of total DNA present in said sample using at    least one other region that is not differently methylated between    said species of DNA and the DNA not originating from cells of said    type, the modification of which region(s) by said reagent is    insensitive to methylation of DNA,    wherein, said detection in step (b) and said detection in step (c)    are made using the same aliquot of DNA of said sample, and in the    same vessel, and effectively simultaneously for such DMRs and other    region(s), and using: (x) the same detectable labels(s) for each of    said DMRs; and (y) a different detectable label(s) for said other    region(s). Terms as set forth herein are generally to be understood    by their common meaning unless indicated otherwise. Where the term    “comprising” or “comprising of” is used herein, it does not exclude    other elements. For the purposes of the present invention, the term    “consisting of” is considered to be a particular embodiment of the    term “comprising of”. If hereinafter a group is defined to comprise    at least a certain number of embodiments, this is also to be    understood to disclose a group that consists of all and/or only of    these embodiments. Where used herein, “and/or” is to be taken as    specific disclosure of each of the two specified features or    components with or without the other. For example “A and/or B” is to    be taken as specific disclosure of each of (i) A, (ii) B and (iii) A    and B, just as if each is set out individually herein. In the    context of the present invention, the terms “about” and    “approximately” denote an interval of accuracy that the person    skilled in the art will understand to still ensure the technical    effect of the feature in question. The term typically indicates    deviation from the indicated numerical value by ±20%, ±15%, ±10%,    and for example ±5%. As will be appreciated by the person of    ordinary skill, the specific such deviation for a numerical value    for a given technical effect will depend on the nature of the    technical effect. For example, a natural or biological technical    effect may generally have a larger such deviation than one for a    man-made or engineering technical effect. Where an indefinite or    definite article is used when referring to a singular noun, e.g.    “a”, “an” or “the”, this includes a plural of that noun unless    something else is specifically stated.

In certain embodiments of the present invention, the individual is ahuman or a non-human animal, where such non-human animal may, inparticular embodiments, be selected from the group consisting of: horse,sheep, cow, pig, chicken, mouse and rat. In a more specific embodiment,the individual is a pregnant female human or a human individualsuspected of being at increased risk of developing or suffering (orsuffering from) a medical condition, such as one or more of the medicalconditions disclosed herein. Such a method of the present invention isnot intended to be practiced on the human or animal body; for example itis intended to be practiced in an in-vitro manner.

In all aspects of the invention, the cell(s) of a given type may be acell of a particular organ or tissues of the same individual. Forexample, the cell may be a tumour cell of the individual. Alternatively,such cell(s) may originate from a different individual or organism. Forexample, in the case of an individual being a pregnant female, the cellof a given type may be a cell of the foetus, including of the placentaof such foetus, and in other embodiments, the cell type may be aninfectious agents such as a bacteria or a protozoa.

In certain embodiments of the present invention, said species of DNAand/or said differently methylated DNA is cell-free DNA, and inparticular of such embodiments is circulating cell-free DNA. In oneparticular embodiment, said species of DNA and the differentlymethylated DNA that is admixed therewith are both circulating cell-freeDNA. The term “cell-free DNA” (or “cfDNA”) is art recognised, andincludes the meaning of DNA that is found outside of a cell, such as ina biological fluid (eg blood, or a blood fraction) of an individual.“Circulating” is also an art-recognised term, and includes the meaningthat an entity or substance (eg cfDNA) is present in, detected oridentified in, or isolated from, a circulatory system of the individual,such as the blood system or the lymphatic system. In particular, whencfDNA is “circulating” it is not located in a cell, and hence may bepresent in the plasma or serum of blood, or it may be present in thelymph of lymphatic fluid.

The term “differentially methylated region” or “DMR” will be recognisedby the person of ordinary skill in the art, and is also intended torefer to a region in chromosomal DNA that is differentially methylated(eg at a CpG motif) between said species of DNA and the other DNA withwhich it is admixed in the sample. For example in one embodiment, theDMRs used in the present invention are differentially methylated betweenfoetal and maternal DNA, or are differentially methylated betweentumour-derived and non-tumour-derived DNA from the same individual. Inparticular embodiments of the present invention, the DMRs arehypermethlyated in foetal DNA and hypo methylated in maternal DNA, orare hypermethylated in tumour-derived DNA and hypomethylated in DNA thatis derived from non-tumour tissue of the individual. That is, in suchregions exhibit a greater degree (ie more) methylation in said speciesof DNA (eg the foetal or tumour cfDNA) as compared to the other DNA (egmaternal or non-tumour DNA), such as about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% or 100% of, or more of, the sites available formethylation at a given DMR are methylated in said species of DNA ascompared to the same sites in the other DNA.

A reagent is used in the present invention that differentially (egselectively) modifies methylated as compared to non-methylated DNA. Forexample, treatment of DNA with a reagent comprising bisulphite(bisulfite) converts cytosine residues to uracil, but leaves5-methylcytosine residues unaffected. Thus, bisulphite treatmentintroduces specific changes in the DNA sequence that depend on themethylation status of individual cytosine residues, yieldingsingle-nucleotide resolution information about the methylation status ofa segment of DNA. Various analyses can be performed on the alteredsequence to retrieve this information, including the use of PCR primersand/or probes that can distinguish between such singe-nucleotidechanges.

Such a reagent may alternatively (or in addition) comprise a restrictionenzyme that is sensitive to the DNA methylation states. Cleavage of sucha restriction enzyme's recognition sequence may be blocked, or impaired,when a particular base in the enzyme's recognition site is modified, egmethylated. In particular embodiments of all aspects of the invention,the reagent comprises a methylation-sensitive restriction enzyme, suchas a methylation-sensitive restriction enzyme disclosed herein;including such embodiments that comprise two, three, four, five or moreof such methylation-sensitive restriction enzymes.

Prior to step (a), the sample may be processed to isolate, enrich and/orpurify, the DNA present therein. For example, a plasma sample may beprocessed using a cfDNA isolation process or kit to provide a(non-natural) subsequent solution that comprises an admixture of saidspecies of DNA together with the differentially methylated DNA that doesnot originate from the cell-type. The step of treating in (a) maycomprise the step of adding a separate solution that comprises saidreagent (eg a methylation sensitive restriction enzyme) to the admixedDNA of the sample (eg, to a non-natural solution comprising such admixedDNA); and/or may comprise maintaining (or changing to) certainconditions. In particular, when said reagent comprises one or moremethylation sensitive restriction enzyme, the step of treating in (a)may comprise incubating the DNA and the enzyme(s) together at about 37°C. for between about 5 min and 300 min, such as between about 30 min and90 min or about 60 min, and optionally may comprise a step of incubatingsuch mixture at a higher temperature (for example, between about 50° C.and 90° C., such as about 80° C.) so as to deactivate the enzyme(s). Incertain embodiments, the composition formed for a treating step of (a)may be non-naturally occurring. For example, particular salts ofcomponents of the solution (or buffer); and/or the mixture of (eg human)cfDNA together with one or more bacterial-derived restriction enzymes(or a non-natural mutant thereof) may be a non-natural composition ormixture. Furthermore, any of the methods of the present invention mayproduce (and hence a composition of the present invention may comprise)an in-vitro-produced nucleic acid molecule, such as a DNA product of aPCR reaction (eg a “PCR product”). One or more of such in-vitro-producednucleic acid molecules may be non-natural because they comprise anucleotide primer and/or probe that includes at least one detectablelabel, such a nucleic acid molecule having been generated by polymeraseextension (or partial nuclease digestion) of such a labelled primerand/or probe, and hence providing at least a fraction of such nucleicacid molecules that include a detectable label, such that even thoughthe nucleic acid sequence of the nucleic acid molecules may comprise anaturally occurring sequence (or fragment thereof), such anin-vitro-produced nucleic acid molecule is non-natural by virtue of (atleast) the non-natural detectable label that it includes.

In contrast, an “other region” (“OR”) used in the present invention isnot (significantly) differentially methylated between said species ofDNA and other DNA with which it is admixed in the sample. For example,under the conditions and nature of the reagent used, there is nodetectable difference between modification by such reagent at the otherregion of said species of DNA (eg foetal DNA) as compared to the otherregion of the admixed DNA (eg the maternal DNA). Such a non-differencemay be achieved if the other region comprises no sites for methylation,if there is no difference in the degree of methylation if such sites arepresent, or by the use of a reagent that does not recognise any sites ofmethylation present in the other region. Accordingly, in alternativeembodiments of the present invention, the at least one other region usedin step (c) is one for which no (significant) difference in methylationbetween said species of DNA and the other DNA is (or can be) recognisedor detected (or recognisable or detectable) with said reagent.

The other region, used in the present invention (that is not sodifferentially methylated) should be (particularly in the context of thefirst aspect of the present invention) or may be (particularly in thecontext of the second aspect of the present invention) non-overlappingwith the DMR used in the present invention. For example, the otherregion, particularly when used in the context of the first aspect of thepresent invention, can be located further than about 10 bp, 20 bp, 50bp, or more than 100 bp, 500 bp, 1 kb or 10 kb, away from the DMR, suchas is located between about 20 bp and about 20 kb upstream or downstreamof (including embodiments being located within the same gene as) saidDMR. In particular, the genomic location of the other region used in thefirst aspect of the present invention is generally located in the sameportion of the genome, such as between about 20 bp and about 20 kbupstream or downstream of (including embodiments being located withinthe same gene as) the genomic location of at least one of the DMRs usedherein. The inventors find that, particularly in the context of thefirst aspect of the present invention, detection (and particularlyquantification) of the species of DNA is enhanced (eg, in terms ofsensitivity, accuracy and/or precision) if the other region is solocated in the same portion of the genome as one of the DMRs. Withoutbeing bound by theory, it is believed that with such similarly-locatedDMR(s) and other region, particularly when used in the context of thefirst aspect of the present invention, the effect of variation inchromatin/nucleosome packing across the genome—and hencestability/degradation of different regions of genomic DNA—is mitigated,such that any difference in stability/degradation of a DMR (ie detectingthe species of DNA) as compared to the other region (is detecting totalDNA) is less, and hence a relative (and absolute) quantification may bemade without it being (significantly) confounded by quantitativedifferences brought about by (significantly) differentialchromatin/nucleosome packing across the genome between a DMR and another region.

In one embodiment of the present invention, the detection of the variousDNA regions, ie the DMR(s) and the other region(s), may occur in asimplified process. Correspondingly, one feature of the presentinvention is that the detection of the various DNA regions, ie the DMRsand the other region(s), occurs in a simplified process. For example,using a single aliquot of DNA from the sample, such DNA regions may bedetected in a single vessel. This feature may simplify the method(s),and can provide for more efficient and accurate detection (especially inthose embodiments when detection is quantitative). The term “vessel”will be art recognised, and includes embodiments of a vessel (such as atube, well of a microtitre plate, nano-well, capillary reaction vesseletc) in which a process or procedure comprised in the method occurs,such as a reaction and/or detection process or a step of a method of thepresent invention. Other such vessels may include droplets in oil/wateremulsions, nanoparticles or a hybridisation chamber; as appropriate tothe detection technology used. The detectable labels used, in certainembodiments of the first aspect of the present invention may be the samefor each DMR and/or may be the same for each other region, providedthat, when detected essentially simultaneously, the label(s) used forthe other region(s) is different (ie, can be separately detected) to thelabel(s) used for the DMR(s). Alternatively, the detectable labels used,in certain embodiments of the first aspect of the present invention maybe different (eg, not the same) for each DMR (or, in respect of certainembodiments of the second aspect of the present invention may be thesame for each set of two or more DMRs) and/or may be different (eg, notthe same) for each other region. The detectable labels used in themethod of the second aspect is the same for each DMR and, in certainembodiments, is the same for each other region, provided that thelabel(s) used for the other region(s) is different (ie, can beseparately detected) to the label(s) used for the DMRs. Detectablelabels that are “the same”, can also include labels while structurallydifferent, are functionally (essentially) similar as they cannot besignificantly differentiated by the detection technology employed. Forexample, structurally different fluorescent dyes may be considered “thesame” if their excitation and emission spectra are (substantially oressentially) similar, or overlap to such a degree that they are able tobe excited and detected simultaneously with the same wavelength(s).Suitable labels (and detection modalities) are further describedelsewhere herein. Preferably, the detection of the DMR(s) and otherregion(s) may be made effectively simultaneously. For example, withinthe same (reaction/detection) vessel, all such regions (and hence saidspecies of DNA and total DNA) can be detected within less than about 5s, 1 s, 0.5 s, 100 ms, 10 ms, 1 ms, 100 us, 10 us or 1 us of each other,and for example without transferring the vessel, or thereaction/mixture, to any subsequent vessel, assay or equipment, or forexample, without adapting or recalibrating the detection process for(any/either of) the DMR(s) or the other region(s) separately. The use of(at least) two different detectable label(s)—one for said DMR(s) and onefor the other region(s)—utilises components, process and/or steps thatare non-natural. For example, a composition of two specific labelstogether with the specific DNA regions would (generally) not be found innature. In particular, short probes used in quantitative probe-basedPCR, while may comprise a DNA sequence that is a fragment of that foundin a natural genome, when linked to a one or more labels (such as afluorescent dye) form a specific labelled fragment that is non-natural.

Collectively, the features of the present invention provide for certainadvantages over prior art methods. These can include sensitive detectionof methylation (and hence the species of DNA to be detected) and/oraccurate and/or improved precision quantification of the amount of saidspecies of DNA by reference to the amount of total DNA detected: (1) inthe first aspect of the present invention, using a co-located otherregion, and optionally within the same assay, from the same aliquot ofadmixed DNA and effectively simultaneously with the detection of the twoor more DMRs; and/or (2) in the second aspect of the present invention,within the same assay, from the same aliquot of admixed DNA andeffectively simultaneously with the detection of the two or more DMRs,and optionally using a co-located other region.

By way of graphical description, a schematic representation of thegeneral arrangement of the DMR(s), the other region(s) and thedetectable label(s), as used for the first aspect of the presentinvention, is presented in FIG. 1(a). (1) The presence of methylation inDNA at DMR1 is detected in the context of an other region (“OR1”) islocated within the same portion of the genome (eg, between about 20 bpand about 20 kb upstream or downstream of) DMR1. (2) Optionally,additional DMRs and/or ORs (such as DMR2 and/or OR2, and up to DMRn andORn) may be detected, and pairs of such additional DMRs and ORs may eachbe co-located in the same portion of the genome (eg, between about 20 bpand about 20 kb upstream or downstream of) as each other. Optionally,(3) the presence of methylation in DNA is detected at multiple DMRs,using the same detectable label(s) and/or (4) the amount of total DNAdetected using at least one OR (OR1, and optionally, OR2 or up to ORn)is detected using different detectable label(s) to those used to detectmethylation at the DMR(s) (optionally, the detectable label(s) used isthe same for all the ORs).

In particular embodiments of the first aspect of the present invention,said detection in step (b) comprises the use of two or more of saidDMRs, and such two or more DMRs may be detected in such step using thesame detectable label(s) for each of said DMRs, or using detectablelabels that are not the same for each of said DMRs. The combination of afeature of the first aspect of the present invention (similarly-locatedDMR(s) and other region(s)) with one of more other feature of thepresent invention: eg the use of at least two DMRs, and/or the detectionin step (b) and the detection in step (c) are made using the samealiquot of DNA of the sample, and in the same reaction/detection vessel,and effectively simultaneously for such DMRs and other region, and/orusing: (x) the same detectable labels(s) for each of said DMRs; and/or(y) a different detectable label for said other region(s); is each apreferred embodiment of the present invention. The use of such acombination of features in the present invention provides opportunityfor efficiency improvements and/or synergistic enchantment of outcome.For example, an improved sensitivity and/or accuracy and/or precision ofdetection (eg, detection of a quantitative amount) or said species ofDNA can be obtained by the use of such a combination; the degree ofimprovement of which can be synergistic, as compared to the use of eachfeature alone; eg the enhancement obtained by use of the combinedfeatures being greater than the sum of each enhancement obtained by theuse of each feature individually.

By way of graphical description, a schematic representation of thegeneral arrangement of the DMRs, the other region(s) and the detectablelabel(s), as used for the second aspect of the present invention, ispresented in FIG. 1(b). (1) The presence of methylation in DNA at two ormore DMRs, DMR1 and DMR2 (and, optionally, up to DMRn), is in each casedetected using the same detectable label(s). (2) Optionally, an otherregion (“OR”) is located within the same portion of the genome (eg,between about 20 bp and about 20 kb upstream or downstream of) one ofthe DMRs). (3) The amount of total DNA detected using at least one OR(OR1, and optionally, OR2 or up to ORn) is detected using differentdetectable label(s) to those used to detect methylation at the DMRs(optionally, the detectable label(s) used is the same for all the ORs).(4) Optionally, methylation at more than two DMRs is so detected, and/orthe amount of total DNA is detected at more than one OR.

In certain embodiments, prior to or as part of the detection that occursas part of a step (b) and/or a step (c) of any method of presentinvention, each DNA region comprising said DMR(s) and/or said otherregion(s), respectively, is(are) amplified. Amplification of DNA may beconducted using any suitable replication process, and in particular ofsuch embodiments, each of the DMR(s) and/or other region(s), isamplified by a polymerase chain reaction (PCR) using primers suitablydesigned for each DMR and/or other region. The person of ordinary skillwill readily be able to design such PCR primers for use in the method ofthe invention, for example by use of primer design algorithms andprograms such as Clone Manager Professional 9 (Sci-Ed Software), VectorNTI (Life Technologies), or web-based tools such as those found fromwww.ncbi.nlm.nih.gov/tools/primer-blast/or molbiol-tools.ca/PCR.htm.Those embodiments of the present invention that comprise PCRamplification can further comprises specific steps that are related tothe practice of PCR, such as any of those described herein, or inparticular the steps of: (A) providing a reaction mixture comprising adouble-stranded target DNA, a pair of primers (for example, a pair ofprimers disclosed herein) designed to amplify a region of such DNA (suchas a DMR or an other region as described herein) wherein the firstprimer is complementary to a sequence on the first strand of the targetDNA and the second primer is complementary to a sequence on the secondstrand of the target DNA, Taq polymerase, and a plurality of freenucleotides comprising adenine, thymine, cytosine and guanine; (B)heating the reaction mixture to a first predetermined temperature for afirst predetermined time to separate the strands of the target DNA fromeach other; (C) cooling the reaction mixture to a second predeterminedtemperature for a second predetermined time under conditions to allowthe first and second primers to hybridise with their complementarysequences on the first and second strands of the target DNA, and toallow the Taq polymerase to extend the primers; and (D) repeating steps(B) and (C) at least 20 times.

In other embodiments, a detectable label used in step (b) and/or step(c) of a method of the invention is independently selected from thegroup consisting of: fluorescent, protein, small molecule or radioactivelabel. For example, fluorescent labels that are the same (including, byhaving similar or overlapping excitation and/or emission spectra) may beused for the DMR(s), and a fluorescent label that has an excitationand/or emission spectra (in particular, a different emission spectrum)may be used for detection of the other region(s). The person of ordinaryskill will be able to select appropriate such fluorescent label(s) foruse in the present invention from, for example, the group consisting of:FAM, TET, JOE, VIC, HEX, NED, PET, ROX, TAMRA, Quasar and Texas Red. Inother embodiments, a detectable label may be a protein or small moleculetag that, for example, can be detected using a specific antibody andELISA-type detection approaches. The use of the same protein or smallmolecule for each of the DMR(s), and a detectably different protein orsmall molecule for the other region(s), may also be utilised for thedetectable label(s) used in the present invention. Different radioactivelabels may be distinguished by their emission energy,penetration/excitation characteristics and particle-type (for example,by distinguishing between alpha and beta particles). Other detectablelabels (such as nucleic-acid coded tag) may also be employed in thepresent invention.

In particular embodiments of the present invention, the detection instep (b) of a method of the example comprises real-time quantitativeprobe-based PCR, eg by using at least one labelled probe which isspecific for one of the DMR(s). In those embodiments where PCRamplification of multiple DMRs is made in the same reaction, such PCRcan be considered as “multiplex” (or “duplex” if only two DMRs are soamplified). Likewise, the detection in step (c) in the methods of theinvention may, in addition or alternatively, comprise real-timequantitative probe-based PCR, such as by using at least one labelledprobe specific for one of said other region(s). In particularembodiments of the second aspect of the present invention, the detectionin step (b) of a method of the example comprises real-time quantitativeprobe-based PCR, eg by using at least two labelled probes, each of whichis specific for one of said DMRs.

The term “probe-based” quantitative PCR is art recognised, andencompasses various embodiments described and marketed under differentbrand names (such as “TaqMan” PCR of Roche), and uses a (eg fluorescent)reporter probe that is specific for the detection of a given amplicon(eg a DMR or an other region). Probe-based quantitative PCR is distinctfrom quantitative PCR using double-stranded DNA-binding dyes (eg SYBRGreen) as reporters, as such double-stranded DNA-binding dyes bindnon-specially to any double-stranded amplicon and eg cannot be used todistinguish between detection of the DMR(s) (ie said species of DNA)from detection of the other region(s) (ie detection of total DNA). Asthe person of ordinary skill will appreciate, a specific amplicon of PCRmay be detected using a single probe or by using multiple probes (suchas two or three probes) for an amplicon. In particular, probe-basedquantitative PCR can include amplification reactions into which havebeen incorporated processes of detecting a target nucleic acid usinglabelled oligonucleotides that use the 5′ to 3′ nuclease activity of anucleic acid polymerase to cleave annealed labelled oligonucleotide (egthe probe) from hybridised duplexes and release labelled oligonucleotidefragments for detection. Such approaches and processes are known in theart and are described in more general terms by Gelfand et al (U55804375,EP0543942 and related family members) and/or Livak et al (U.S. Pat. No.6,258,569, EP0792374 and related family members), and include where theprobe comprises a detectable label in combination with a quenchermolecule that quenches the detectability of the label when bound, suchthat 5′ to 3′ nuclease (and hence amplification) is required to occurbefore the detectable label is released into the reaction mixture (wayfrom the quencher) and hence may be detected. Furthermore, “probe-based”quantitative PCR approaches may by alternatively or additionallyenhanced by the use of probes that comprise anoligonucleotide-fluorophore-quencher-minor groove binder conjugates,such as described by Reed et al (U.S. Pat. No. 6,727,356, EP1235938 andrelated family members).

Such probe-based quantitative PCR may be conducted in ananalogue-approach, using a machine such as a LightCycler in which theintensity of signal (eg over time) is measured and used toquantitatively determine detection. Systems and approaches for suchdetection are described by Woudenberg et al (U56929907, EP0706649 andrelated family members) and/or Higuchi (U55994056, EP0512334 and relatedfamily members). Alternatively, digital PCR (dPCR), ie, PCR conducted inmultiple events so as to determine the number of amplification events asmethod to quantitate an amount of detected DNA. For example, dPCR thatis conducted in nano-wells or droplets (ddPCR).

The person of ordinary skill will be able to design suitable primers andprobes (and with suitable labels, eg dyes) for probe-based quantitativePCR detection of the DMRs and/or other regions(s); for example by usingprimer/probe design software as described elsewhere herein. As will beknown, the PCR primers may overlap methylation site(s) specific for themethylation-specific modifying reagent used in the methods, inparticular when the reagent comprises one or more methylation sensitiverestriction enzyme, such as one (or a combination thereof) as disclosedherein. In particular such embodiments, one or other (or when consideredtogether, both) of the PCR primers for a given DMR may overlap two orthree such methylation sites (such as two or three restriction sites formethylation-sensitive restriction enzymes, each of which may comprise,or comprises, a methylation site). Alternatively or in addition, theprimers for a DMR may be designed to flank one, two, three or more suchmethylation sites, such as up to 10, 15, 20, 25 or 50 such methylationsites, in particular flanking restriction sites for one, two, three ormore such methylation sites, such as up to 10, 15, 20, 25 or 50methylation-sensitive restriction enzymes, each of which may comprise,or comprises, a methylation site.

In a particular embodiment of the second aspect of the invention, thegenomic location of the other region, when used in such aspect isgenerally located in the same portion of the genome, such as betweenabout 20 bp and about 20 kb upstream or downstream of (includingembodiments within the same gene as) the genomic location of at leastone of the DMRs used herein. In certain embodiments of such aspect, theother region, when used in the second aspect of the present invention,does not overlap with the DMR. The inventors find that, in the secondaspect of the present invention, detection (and particularlyquantification) of the species of DNA is enhanced (eg, in terms ofsensitivity, accuracy and/or precision) if the other region is solocated in the same portion of the genome as one of the DMRs. Withoutbeing bound by theory, it is believed that with such similarly-locatedDMR(s) and other region when used in the second aspect of the presentinvention, the effect of variation in chromatin/nucleosome packingacross the genome—and hence stability/degradation of different regionsof genomic DNA—is mitigated, such that any difference instability/degradation of a DMR (ie detecting the species of DNA) ascompared to the other region (is detecting total DNA) is less, and hencea relative (and absolute) quantification may be made without it being(significantly) confounded by quantitative differences brought about by(significantly) differential chromatin/nucleosome packing across thegenome between a DMR and an other region. The combination of thisfeature (similarly-located DMR(s) and other region) with another featureof the present invention (the use of at least two DMRs, and thedetection in step (b) and the detection in step (c) are made using thesame aliquot of DNA of the sample, and in the same reaction/detectionvessel, and effectively simultaneously for such DMRs and other region,and using: (x) the same detectable labels(s) for each of said DMRs; and(y) a different detectable label for said other region(s)), is apreferred embodiment of the present invention. The use of such acombination of features in the present invention provides opportunityfor efficiency improvements and/or synergistic enchantment of outcome.For example, an improved sensitivity and/or accuracy and/or precision ofdetection (eg, detection of a quantitative amount) or said species ofDNA can be obtained by the use of such a combination; the degree ofimprovement of which can be synergistic, as compared to the use of eachfeature alone; eg the enhancement obtained by use of the combinedfeatures being greater than the sum of each enhancement obtained by theuse of each feature individually.

The present invention includes the use of one other region to providefor the detection of an amount of total DNA in the admixture. However,the present invention also encompasses embodiments that use more thanone other region. For example, the invention includes such embodimentswherein said detection in step (c) comprises using at least two of saidother regions, such as two, three or four of said other regions. Inparticular embodiments of all aspects of the present invention, thenumber of said other regions is the same as the number of DMRs used instep (b). For example, if two DMRs are used then two other regions areused in such an embodiment, and if three DMRs are used then three otherregions are used (such as depicted in FIG. 1).

As described elsewhere herein, the first aspect of present inventionincludes where the other region is generally located in the same portionof the genome, such as between about 20 bp and about 20 kb upstream ordownstream of (including embodiments within the same gene as) thegenomic location of at least one of the DMRs used herein. Also asdescribed elsewhere herein, certain embodiments of the second aspect ofpresent invention include where the other region is generally located inthe same portion of the genome, such as between about 20 bp and about 20kb upstream or downstream of (including embodiments within the same geneas) the genomic location of at least one of the DMRs used herein. Incertain embodiments of such second aspect, the other region does notoverlap with the DMR. Accordingly, if multiple other regions are used inthe present invention, then embodiments are included where two or moreof such other regions are similarly located in the genome to the two ormore DMRs. For example, one of said other regions may be located betweenabout 20 bp and about 20 kb upstream or downstream of (includingembodiments within the same gene as) a DMR used in step (b) and eachother of the said other regions (eg, a second other region) is locatedbetween about 20 bp and about 20 kb upstream or downstream of (includingembodiments within the same gene as) another of said (eg,non-overlapping) DMRs (eg, the second DMR). In certain embodiments anadditional other region, may overlap with a DMR.

An other region used in the present invention, when generally located inthe same portion of the genome as a DMR, may be located upstream ordownstream of one of said DMRs within a distance selected from the groupconsisting of: between about 16 kb to 20 bp, 14 kb to 20 bp, 12 kb to 20bp, 10 kb to 20 bp, 8 kb to 20 bp, 6 kb to 20 bp, 5 kb to 20 bp, 4 kb to20 bp, 3 kb to 2 bp, 16 kb to 20 bp, 1 kb to 20 bp, 500 bp to 20 bp, 200bp to 20 bp, 20 kb to 15 kb, 15 kb to 10 kb, 12 kb to 8 kb, 10 kb to 8kb, 11 kb to 7 kb, 11 kb to 10 kb, 9 kb to 8 kb, 8 kb to 6 kb, 6 kb to 4kb, 4 kb to 2 kb, 2 kb to 500 bp, 1 kb to 100 bp, 500 bp to 50 bp, 400bp to 200 bp and 500 bp to 100 bp and 500 bp to 300 bp. In particularembodiments, each other region used in the present invention is sogenerally located to a different of the DMRs used.

If multiple other regions are used, then the present invention includesembodiments where the detection in step (c) is made using the samedetectable label for each of said other regions and/or comprisesmultiplex real-time quantitative PCR using at least two labelled probeseach of which is specific for one of said other regions.

In particular embodiments, all detection steps (ie, those required forall DMR(s) and all other region(s)) are conducted in an efficient andeffective manner using multiplex quantitative probe-based (eg TaqMan)PCR, in one process step or reaction. For example, the detection in step(c) and said detection in step (b) are made using the same aliquot ofDNA of said sample, and in the same reaction/detection vessel, andeffectively simultaneously with each other, and by multiplex real-timequantitative PCR using at least one labeled probe specific for each ofthe said DMRs and other region(s). In particular of such embodiments,the reagent comprises one or more methylation sensitive restrictionenzyme, such as one (or a combination thereof) as disclosed herein.

The present invention may also include further procedures, such as oneor more control procedures. For example, the present invention caninclude one or more steps directed to the detection of a third class ofDNA region that acts as a control for the modification step (eg, as acontrol for restriction enzyme digestion). Such embodiments may, forexample, also be conducted using multiplex real-time quantitativeprobe-based PCR wherein such control region is amplified and detected bya third set of primer/probe(s) with a third detectable label used forsuch class of region.

In one embodiment of the present invention of particular relevance, saidspecies of DNA originates from cells of a foetus and/or the placenta ofa foetus and said sample is from a pregnant female. In such embodiments,the sample may be obtained in a non-invasive manner. For example, saidspecies of DNA is circulating cell-free DNA that has been detected fromthe sample being blood or a blood fraction (such as plasma or serum)that has been obtained from the pregnant female by conventional meanssuch as a blood collection tube. In such embodiments, the sample willcomprise DNA (such as said other DNA) that has a maternal origin; thatis it originates from cells (and hence the genome of) the pregnantfemale.

The present invention includes embodiments where the DMR(s) is(are)hypermethlyated in foetal DNA and hypo methylated in maternal DNA. Incertain embodiments, such a DMR may be located in a promoter, enhancerregion or an exon of a gene, such as a gene disclosed herein.Alternatively, a DMR may be located in an intron of such a gene, or in anon-coding region of the genome. In particular embodiments of allaspects of the present invention, such genome and/or gene is a humangenome or gene. Specifically included in the present invention areembodiments wherein said DMR(s) comprises at least one, preferably atleast two, methylation site(s) specific for said reagent, and at leastone of said DMR(s) is located in a portion of the genome and/or gene (ega human genome or gene) that is RASSF1A and/or TBX3, or selected fromthe group consisting of: RASSF1A, TBX3, HLCS, ZFY, CDC42EP1, MGC15523,SOX14 and SPN and DSCAM. Also, embodiments are included wherein saidDMR(s) comprises at least one, preferably at least two, methylationsite(s) specific for said reagent, and at least one of said DMR(s) islocated in a region and/or gene selected from the group consisting of:AIRE, SIM2, ERG, VAPA-APCDDI, one disclosed in WO 2011/034631 as beinghypermethylated in foetal DNA relative to maternal DNA (eg, SEQ ID NOs:1-59, 90-163, 176, 179, 180, 184, 188, 189, 190, 191, 193, 195, 198,199, 200, 201, 202, 203, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 221, 223, 225, 226, 231, 232, 233, 235, 239, 241, 257, 258, 259,and/or 261 of WO 2011/034631) and one disclosed in WO 2011/092592 (eg,EP1, EP2, EP3, EP4, EP5, EP6, EP7, EP8, EP9, EP10, EP11 and/or EP12 ofWO 2011/092592 (SEQ ID NOs: 33-44 of WO 2011/092592), as furtherinvestigated in Lim et al 2014, BMC Medical Genomics 7:1). TABLE A showsthe conversion of the sequence identifiers used in the WO 2011/034631and WO 2011/092592 to the sequence identifiers used in the presentinvention.

TABLE A Conversion table for sequence indentifiers SEQ ID NO.: SEQ IDNO.: SEQ ID NO.: Present invention WO 2011/034631 WO 2011/092592 SEQ IDNO.: 15 1 — SEQ ID NO.: 16 2 — SEQ ID NO.: 17 3 — SEQ ID NO.: 18 4 — SEQID NO.: 19 5 — SEQ ID NO.: 20 6 — SEQ ID NO.: 21 7 — SEQ ID NO.: 22 8 —SEQ ID NO.: 23 9 — SEQ ID NO.: 24 10 — SEQ ID NO.: 25 11 — SEQ ID NO.:26 12 — SEQ ID NO.: 27 13 — SEQ ID NO.: 28 14 — SEQ ID NO.: 29 15 — SEQID NO.: 30 16 — SEQ ID NO.: 31 17 — SEQ ID NO.: 32 18 — SEQ ID NO.: 3319 — SEQ ID NO.: 34 20 — SEQ ID NO.: 35 21 — SEQ ID NO.: 36 22 — SEQ IDNO.: 37 23 — SEQ ID NO.: 38 24 — SEQ ID NO.: 39 25 — SEQ ID NO.: 40 26 —SEQ ID NO.: 41 27 — SEQ ID NO.: 42 28 — SEQ ID NO.: 43 29 — SEQ ID NO.:44 30 — SEQ ID NO.: 45 31 — SEQ ID NO.: 46 32 — SEQ ID NO.: 47 33 — SEQID NO.: 48 34 — SEQ ID NO.: 49 35 — SEQ ID NO.: 50 36 — SEQ ID NO.: 5137 — SEQ ID NO.: 52 38 — SEQ ID NO.: 53 39 — SEQ ID NO.: 54 40 — SEQ IDNO.: 55 41 — SEQ ID NO.: 56 42 — SEQ ID NO.: 57 43 — SEQ ID NO.: 58 44 —SEQ ID NO.: 59 45 — SEQ ID NO.: 60 46 — SEQ ID NO.: 61 47 — SEQ ID NO.:62 48 — SEQ ID NO.: 63 49 — SEQ ID NO.: 64 50 — SEQ ID NO.: 65 51 — SEQID NO.: 66 52 — SEQ ID NO.: 67 53 — SEQ ID NO.: 68 54 — SEQ ID NO.: 6955 — SEQ ID NO.: 70 56 — SEQ ID NO.: 71 57 — SEQ ID NO.: 72 58 — SEQ IDNO.: 73 59 — SEQ ID NO.: 74 90 — SEQ ID NO.: 75 91 — SEQ ID NO.: 76 92 —SEQ ID NO.: 77 93 — SEQ ID NO.: 78 94 — SEQ ID NO.: 79 95 — SEQ ID NO.:80 96 — SEQ ID NO.: 81 97 — SEQ ID NO.: 82 98 — SEQ ID NO.: 83 99 — SEQID NO.: 84 100 — SEQ ID NO.: 85 101 — SEQ ID NO.: 86 102 — SEQ ID NO.:87 103 — SEQ ID NO.: 88 104 — SEQ ID NO.: 89 105 — SEQ ID NO.: 90 106 —SEQ ID NO.: 91 107 — SEQ ID NO.: 92 108 — SEQ ID NO.: 93 109 — SEQ IDNO.: 94 110 — SEQ ID NO.: 95 111 — SEQ ID NO.: 96 112 — SEQ ID NO.: 97113 — SEQ ID NO.: 98 114 — SEQ ID NO.: 99 115 — SEQ ID NO.: 100 116 —SEQ ID NO.: 101 117 — SEQ ID NO.: 102 118 — SEQ ID NO.: 103 119 — SEQ IDNO.: 104 120 — SEQ ID NO.: 105 121 — SEQ ID NO.: 106 122 — SEQ ID NO.:107 123 — SEQ ID NO.: 108 124 — SEQ ID NO.: 109 125 — SEQ ID NO.: 110126 — SEQ ID NO.: 111 127 — SEQ ID NO.: 112 128 — SEQ ID NO.: 113 129 —SEQ ID NO.: 114 130 — SEQ ID NO.: 115 131 — SEQ ID NO.: 116 132 — SEQ IDNO.: 117 133 — SEQ ID NO.: 118 134 — SEQ ID NO.: 119 135 — SEQ ID NO.:120 136 — SEQ ID NO.: 121 137 — SEQ ID NO.: 122 138 — SEQ ID NO.: 123139 — SEQ ID NO.: 124 140 — SEQ ID NO.: 125 141 — SEQ ID NO.: 126 142 —SEQ ID NO.: 127 143 — SEQ ID NO.: 128 144 — SEQ ID NO.: 129 145 — SEQ IDNO.: 130 146 — SEQ ID NO.: 131 147 — SEQ ID NO.: 132 148 — SEQ ID NO.:133 149 — SEQ ID NO.: 134 150 — SEQ ID NO.: 135 151 — SEQ ID NO.: 136152 — SEQ ID NO.: 137 153 — SEQ ID NO.: 138 154 — SEQ ID NO.: 139 155 —SEQ ID NO.: 140 156 — SEQ ID NO.: 141 157 — SEQ ID NO.: 142 158 — SEQ IDNO.: 143 159 — SEQ ID NO.: 144 160 — SEQ ID NO.: 145 161 — SEQ ID NO.:146 162 — SEQ ID NO.: 147 163 — SEQ ID NO.: 148 176 — SEQ ID NO.: 149179 — SEQ ID NO.: 150 180 — SEQ ID NO.: 151 184 — SEQ ID NO.: 152 188 —SEQ ID NO.: 153 189 — SEQ ID NO.: 154 190 — SEQ ID NO.: 155 191 — SEQ IDNO.: 156 193 — SEQ ID NO.: 157 195 — SEQ ID NO.: 158 198 — SEQ ID NO.:159 199 — SEQ ID NO.: 160 200 — SEQ ID NO.: 161 201 — SEQ ID NO.: 162202 — SEQ ID NO.: 163 203 — SEQ ID NO.: 164 205 — SEQ ID NO.: 165 206 —SEQ ID NO.: 166 207 — SEQ ID NO.: 167 208 — SEQ ID NO.: 168 209 — SEQ IDNO.: 169 210 — SEQ ID NO.: 170 211 — SEQ ID NO.: 171 212 — SEQ ID NO.:172 213 — SEQ ID NO.: 173 214 — SEQ ID NO.: 174 221 — SEQ ID NO.: 175223 — SEQ ID NO.: 176 225 — SEQ ID NO.: 177 226 — SEQ ID NO.: 178 231 —SEQ ID NO.: 179 232 — SEQ ID NO.: 180 233 — SEQ ID NO.: 181 235 — SEQ IDNO.: 182 239 — SEQ ID NO.: 183 241 — SEQ ID NO.: 184 257 — SEQ ID NO.:185 258 — SEQ ID NO.: 186 259 — SEQ ID NO.: 187 261 — SEQ ID NO.: 188 —33 SEQ ID NO.: 189 — 34 SEQ ID NO.: 190 — 35 SEQ ID NO.: 191 — 36 SEQ IDNO.: 192 — 37 SEQ ID NO.: 193 — 38 SEQ ID NO.: 194 — 39 SEQ ID NO.: 195— 40 SEQ ID NO.: 196 — 41 SEQ ID NO.: 197 — 42 SEQ ID NO.: 198 — 43 SEQID NO.: 199 — 44

In other embodiments of the present invention, at least one of saidDMR(s) is located on a human chromosome selected from the listconsisting of: chromosome 21, chromosome 18, chromosome 13, X-chromosomeand Y-chromosome, preferably at least one of said DMR(s) is located onchromosome 21, chromosome 18 or chromosome 13, more preferably at leastone of said DMR(s) is located on chromosome 21. In other or furtherembodiments of the present invention, at least one of said DMR(s)comprises at least one, preferably at least two, methylation site(s)specific for said reagent, and said DMR is located in a region and/orgene selected from the list consisting of: maspin (and preferably aportion of the maspin (aka “SERPINB5”) gene that is described inEP1751307 as being differentially methylated between a foetus and itsmother), CGI137, PDE9A, PPP1R2P2, Similarity to Fem1A (C. elegans),CGI009, CBR1, DSCAM, C21orf29 and CGI13 (or such as a gene and/or regiondisclosed in Table 1 of WO 2007/132167 or in Chim et al 2008 that areunmethylated in maternal blood cells and methylated in placenta).

Human chromosome 21, chromosome 18, chromosome 13 and X-chromosome arethose that are generally considered to be associated with chromosomalaneuploidy (particularly of a foetus), and using at least one DMR thatis located in genes or regions of such chromosomes are particularlypreferred for those methods of the present invention for the detection,identification or quantification of a species of DNA that is associatedwith a chromosomal aneuploidy and/or for the diagnosis of chromosomalaneuploidy (particularly of a foetus) such as a chromosomal trisomyincluding trisomy of chromosome 21 (also known as Down's Syndrome).Accordingly, in certain embodiments of the present invention, at leastone of said DMR(s) is located in a region and/or gene selected from thelist consisting of: SEQ ID NOs 1-39, 176, 179, 180, 184, 188, 189, 190,191, 193, 195, 198, 199, 200, 201, 202, 203, 205, 206, 207, 208, 209,210, 211, 212, 213, 214, 221, 223, 225, 226, 231, 232, 233, 235, 239,241, 257, 258, 259, and/or 261 of WO 2011/034631, preferably selectedfrom the list consisting of: SEQ ID No NOs 33-39, 176, 179, 180, 184,188, 189, 190, 191, 193, 195, 198, 199, 200, 201, 202, 203, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 221, 223, 225, 226, 231, 232,233, 235, 239, 241, 257, 258, 259, and/or 261 of WO 2011/034631, morepreferably selected from the list consisting of: SEQ ID No NOs 184, 191,201, 202, 208, 209, 210, 211, 212, 214, 235, 241 and 258 of WO2011/034631. In other or further embodiments of the present invention,at least one of said DMR(s) comprises at least one, preferably at leasttwo, methylation site(s) specific for said reagent, and at least one ofsaid DMR(s) is located in a region and/or gene disclosed in WO2011/092592, including on selected from the list(s) consisting of: EP1,EP2, EP3, EP4, EP5, EP6, EP7, EP8, EP9, EP10, EP11 and EP12 of WO2011/092592 (SEQ ID NOs: 33-44 of WO 2011/092592). In yet other orfurther embodiments of the present invention, at least one of saidDMR(s) comprises at least one, preferably at least two, methylationsite(s) specific for said reagent, and at least one of said DMR(s) islocated in a region and/or gene selected from the list consisting of:AIRE, SIM2, ERG and VAPA-APCDDI, or is HLCS.

In certain embodiments of the invention, at least one of the said DMRsis located on a human chromosome that is not (or rarely) associated witha chromosomal aneuploidy. In such embodiments, such chromosome can beconsidered as a (diploid) “reference chromosome”, from which a speciesof DNA may be detected, identified or quantified that reflects anestimate of total diploid DNA of eg the foetus relative to maternal DNA.A parameter (such as a relative or absolute amount) in respect of suchdetected, identified or quantified species of DNA (from such a“reference” chromosome) can then be compared to a correspondingparameter in respect of a detected, identified or quantified species ofDNA from a chromosome, or part thereof, associated with a chromosomalaneuploidy (such as trisomy); where any significant difference in suchcompared parameters (such as an excess of one chromosomal amount overanother chromosomal amount) being indicative that a chromosomalaneuploidy may exist. Accordingly, in certain embodiments of theinvention, at least one of the said DMRs is located on a humanchromosome selected from the list consisting of: chromosome 1 to 12,chromosome 14 to 17, chromosome 19, chromosome 20 chromosome 22 andchromosome 23, preferably said DMRs is located on a human chromosome 2,chromosome 3 or chromosome 12; In other or further embodiments of thepresent invention, at least one of said DMR(s) comprises at least one,preferably at least two, methylation site(s) specific for said reagent,and said DMR is located in a regions and/or gene selected from the listconsisting of: CD48, FAIM3, ARHGAP25, SELPLG, APC, CASP8, RARB, SCGB3A1,DAB2IP, PTPN6, THY1, TMEFF2 and PYCARD. In alternative or additionalother or further embodiments of the present invention, at least one ofsaid DMR(s) is located in a region and/or gene selected from the listconsisting of: RASSF1A, TBX3, ZFY, CDC42EP1, MGC15523, SOX14 and SPN;and/or said DMR(s) is located in a region and/or gene selected from thelist consisting of: SEQ ID NOs: 40-59 and 90-163 of WO 2011/034631.

If two DMRs are used, then in particular embodiments of all aspects ofthe present invention, they are not located in the same portion of thegenomic and/or gene. For example, such DMRs may be located on separatechromosomes, or separated by more than about 20 kb, or more than about15 kb, 10 kb, 8 kb, 6 kb, 4 kb, 2 kb, 1 kb, 500 bp or 200 bp.Alternatively, it is envisioned, that when two (or more) DMRs are usedin the present invention, they may, in certain embodiments, be locatedin the same region or gene (such as one described herein) and, further,may overlap with each other.

In particular embodiments of the present invention, a plurality ofspecies of DNA are detected in said sample. For example, two (or more)species of DNA can be detected (identified or quantified) in said sampleusing a method of the invention. Each species of DNA may be on (ororiginate from) separate chromosomes; including a first species of DNAon (or originating from) a chromosome relevant to a chromosomalaneuploidy (for example, human chromosome 21, 18, 13 or X), and a secondspecies of DNA on (or originating from) a reference chromosome (forexample, human chromosome 1 to 12, 14 to 17, 19, 20, 22 or 23. Thedetection, identification or quantification of a first species of DNA on(or originating from) a chromosome relevant to a chromosomal aneuploidy,and of a second species of on (or originating from) a referencechromosome, can enable respective parameters (such as a relative orabsolute amount) from each such detection, identification orquantification to be compared (for example, via relative amount orratio) and hence useful for the detection, identification or diagnosisof a chromosomal aneuploidy, particularly in a foetus.

In particular embodiments of the present invention, when two of saidDMRs are used (or more than two DMRs are being used) each is located ina portion of the genome and/or gene (preferably that is human) that isRASSF1A and/or TBX3, or is selected from the group consisting of:RASSF1A, TBX3, HLCS, ZFY, CDC42EP1, MGC15523, SOX14 and SPN and DSCAM;and/or at least one of said DMRs is located between about positions4,700 bp and 5,600 bp of RASSF1A (NCBI Reference Sequence: NG_023270.1:Homo sapiens Ras association (RalGDS/AF-6) domain family member 1(RASSF1), RefSeqGene on chromosome 3; SEQ ID NO.: 13) or about positions1,660 bp and 2,400 bp of TBX3 (NCBI Reference Sequence: NG_008315.1:Homo sapiens T-box 3 (TBX3), RefSeqGene on chromosome 12; SEQ ID NO.:14) or is located between about positions 40,841,584 and 40,842,020 ofDSCAM; (Down Syndrome Cell Adhesion Molecule; NCBI Reference SequenceHomo sapiens chromosome 21, GRCh38.p2 Primary Assembly: NC_000021.9GI:568815577, region 40,010,999 to 40,847,113; SEQ ID No.: 200). In amore particular embodiment, two (or more) DMRs are used, and a first DMRcomprises one located between about positions 4,700 bp and 5,600 bp ofRASSF1A and a second DMR comprises one located between about positions1,660 bp and 2,400 bp of TBX3.

In particular embodiments, a DMR is located in RASSF1A between aboutpositions 4,900 bp and 5,500 bp, 5,000 bp and 5,400 bp, or 5,100 bp and5,300 bp of RASSF1A; and/or is located in TBX3 between about positions1,800 bp and 2,260 bp, 1,920 bp and 2,160 bp or 1,920 bp and 2,080 bp ofTBX3 (such as SEQ ID No.: 203); and/or is located in DSCAM between aboutpositions 40,841,600 bp and 40,841,900 bp, 40,841,625 bp and 40,841,840bp or 40,841,650 bp and 40,841,790 bp of DSCAM (with reference to Homosapiens chromosome 21, GRCh38.p2 Primary Assembly: NC_000021.9GI:568815577, region), such as SEQ ID No.: 201.

The general arrangement of the DMRs and other regions (“OR”) used in oneembodiment of the present invention, is graphically represented by FIG.2: (1a) DMR1 is found in exon 2 of RASSF1A and OR1 is located withinexon 4 of RASSF1A, with DMR1 located between positions 50,340,672 bp and50,340,784 bp and OR1 located between positions 50,331,604 bp and50,331,702 bp of the RASS1A genomic sequence (NCBI Reference Sequence:NC_000003.12 Homo sapiens chromosome 3, GRCh38 Primary Assembly),separating DMR1 and OR1 by a distance of 8,969 bp. (1b) DMR2 is found inthe promoter region of TBX3, with DMR2 located between positions114,687,095 bp and 114,687,189 bp and OR2 is located between positions114,676,384 bp and 114,676,454 bp of the TBX3 genomic sequence (NCBIReference Sequence: NC_000012.12 Homo sapiens chromosome 12, GRCh38Primary Assembly), separating DMR2 and OR2 by a distance of 10,640 bp.(2) Methylation in DNA at the two DMRs is detected using probe-basedquantitative PCR using the respective forward (F) and reverse (R) PCRprimers and region-specific probes, each probe labelled with the samelabels (P*). (3) Total DNA is detected at two ORs using probe-basedquantitative PCR using the respective forward (F) and reverse (R) PCRprimers and region-specific probes, each probe labelled with the samelabels for the ORs that is different to the labels used for the two DMRs(P**). Details of primer and probe sequences and probe labels are setout in TABLE 1.

The general arrangement of the DMRs and other regions (“OR”) used inanother embodiment of the present invention, is graphically representedby FIG. 6: (1) DMR1 is found in eg DSCAM and OR1 is located in eg DSCAM,with DMR1 located between positions 40,841,691 bp and 40,841,781 bp andOR1 located between positions 40,841,286 bp and 40,841,772 bp of theDSCAM genomic sequence (Down Syndrome Cell Adhesion Molecule; NCBIReference Sequence Homo sapiens chromosome 21, GRCh38.p2 PrimaryAssembly: NC_000021.9 GI:568815577, region 40010999 to 40847113; SEQ IDNo.: 200), separating DMR1 and OR1 by a distance between about 300 bpand 500 bp. (1′) DMR1′ is found in TBX3 and OR1′ is located in TBX3,with DMR1′ located between positions 114,687,093 bp and 114,687,191 bpand OR1′ located between positions 114,676,384 bp and 114,676,454 of theTBX3 genomic sequence (NCBI Reference Sequence: NC_000012.12 Homosapiens chromosome 12, GRCh38 Primary Assembly), separating DMR1′ andOR1′ by a distance between about 10,600 bp and 10,810 bp.

Certain embodiments of the present invention, in the context of themethods, compositions, kits and/or computer program product thereof,comprise or comprise the use of one or more of the forgoing DMRs, ORs,sequences of the primers and/or probes, in particular any of those setforth in TABLE 1 or TABLE 8. In certain of such embodiments, a givenprobe comprises a sequence set forth in TABLE 1 or TABLE 8 and any oneof the label and quencher pairs (optionally, with a minor binding groovemoiety) as set forth in TABLE 1 or TABLE 8. In particular, the probe maycomprise the combination of the sequence with the label and quencherpair (optionally, with the minor binding groove moiety) as set forth inTABLE 1 or TABLE 8 for such probe. Other embodiments of the presentinvention, particularly in the context of the methods, compositions,kits and/or computer program product thereof, comprise or comprise theuse of the specific combination of two or more (for example, of all) theforegoing DMRs, ORs, sequences of the primers and/or probes, inparticular the combination of the primers/probes as set forth in TABLE 1or the combination of the primers/probes as set forth in TABLE 8.

The term “methylation site(s)” will be art-recognised, and has a meaningthat encompasses, for example, a CpG motif within a short nucleotidesequence (eg one that is 4, 6, 8, 10 or 12 bp in length) that is,preferably, recognised by a methylation-sensitive restriction enzyme,such as one disclosed elsewhere herein.

Analogously, and particularly in the context of the first aspect of thepresent invention, the other region, when located in particular portionsand/or genes of the genome, may be located in a promoter, enhancerregion or an exon of a gene, or alternatively, located in an intron ofsuch a gene, or in a non-coding region of the genome. In particularembodiments of all aspects of the present invention, such genome and/orgene is a human genome or gene. In particular embodiments, when an otherregion used in the present invention is located in the same portion ofthe genome and/or gene that features one or more DMRs (preferably,non-overlapping with a DMR used in the invention), then it is located ina portion of the genome and/or gene such as a gene (eg human, and/or inparticular when said species of DNA is foetal cfDNA) that is RASSF1Aand/or TBX3, or is selected from the group consisting of: RASSF1A, TBX3,HLCS, ZFY, CDC42EP1, MGC15523, SOX14 and SPN and DSCAM. When notco-located with a DMR (for example, when a second or multiple otherregion is used), then such other region may, in certain embodiments, belocated in a (eg human) housekeeping gene (such as GAPDH, beta-actin,ALB, APOE or RNASEP). Analogously, and particularly in the context ofthe second aspect of the present invention, the other region may belocated in particular portions and/or genes of the genome, and may belocated in a promoter, enhancer region or an exon of a gene, oralternatively, located in an intron of such a gene, or in a non-codingregion of the genome. In particular embodiments of all aspects of thepresent invention, such genome and/or gene is a human genome or gene. Inparticular embodiments, an other region used in the present invention islocated in a (eg human) housekeeping gene (such as GAPDH, beta-actin,ALB, APOE or RNASEP). Alternatively (and in particular when said speciesof DNA is foetal cfDNA), said other region may be located in the sameportion of the genome and/or gene that feature one or more DMRs (such asthose RASSF1A, TBX3, HLCS, ZFY, CDC42EP1, MGC15523, SOX14 or SPN orDSCAM), and preferably does not overlap with a DMR used in theinvention.

In particular embodiments of all aspects of the invention, said otherregion comprises a portion of the genome without a methylation sitespecific for said reagent, and said other region is located in the (eghuman) genes RASSF1A or TBX3 (eg SEQ ID NOs: 13 and 14 respectively) orDSCAM (SEQ ID No.: 200), and includes more particular embodimentswherein two or more of said other regions are used in detection step (c)and the first other region is located between about positions 14,220 bpand 13,350 bp of such RASSF1A and the second other region is locatedbetween about positions 12,400 bp and 13,000 bp of such TBX3. Inparticular embodiments, an other region is located in RASSF1A betweenabout positions 14,230 bp and 14,340 bp, 14,230 bp and 14,330 bp, 14,230bp and 14,320 bp, or 14,230 bp and 14,310 bp of such RASSF1A; and/or islocated in TBX3 between about positions 12,400 bp and 12,940 bp, 12,700bp and 12,850 bp or 12,710 bp and 12,790 bp of such TBX3 (such as SEQ IDNo.: 204); and/or is located in DSCAM between about positions 40,841,150bp and 40,841,525 bp, 40,841,200 bp and 40,841,475 bp or 40,841,250 bpand 40,841,425 bp of DSCAM (with reference to Homo sapiens chromosome21, GRCh38.p2 Primary Assembly: NC_000021.9 GI:568815577, region), suchas SEQ ID No.: 202. Alternatively, an other region may be located in anexon such as between about positions 13,790 bp and 13,880 bp, or 14,490bp and 14,600 bp of such RASSF1A, or between about positions 8,040 bpand 8,180 bp or 6,230 bp and 6,350 bp of such TBX3; or an other regionmay be located in an intron such as between about positions 10,500 bpand 11.90 bp of such RASSF1A, or between about positions 10,000 bp and11,000 bp of such TBX3

There is now strong evidence that the level of foetal cfDNA (and/ortotal cfDNA) present in the circulatory system (eg in plasma) of apregnant female is a marker of one or more forms of preeclampsia, suchas early-onset preeclampsia, mild and/or severe preeclampsia (see Hahnet al 2011, Placenta 32(Supl):S17). The present invention showsparticular utility in the efficient, effective, sensitive and/orlow-variability detection/quantification of foetal cfDNA present inplasma of pregnant females, and the present invention has particularutility therein. Accordingly, in particular embodiments of the presentinvention, the individual is a pregnant female and is susceptible tosuffering or developing a pregnancy-associated medical condition;particularly where said pregnancy-associated medical condition ispreeclampsia. As used herein, an individual “susceptible to” a medicalcondition may alternatively be described as “is suspected to” or to “beconsidered at risk of being susceptible to” suffering or developing amedical condition; and in certain embodiments, the present invention isused to screen and/or diagnose the individual for susceptibility to,risk of suffering or developing, or suffering from or developing, amedical condition.

In alternative embodiments, the individual is a pregnant female and issusceptible to (or considered at risk of being susceptible to) sufferingor developing a pregnancy-associated medical condition selected from thegroup consisting of: preterm labour, intrauterine growth retardation andvanishing twin. In particular, the inventors were surprised that thesensitivity of the present invention was such that discrepancies betweencfDNA levels determined by the method of the invention and thatdetermined by counts of Y-chromosome sequences as determined bymassively parallel sequencing approaches, was useful in identifying oneor more cases of a vanishing twin in (mixed-sex) twin pregnancies thatpreviously were believed to be singleton pregnancies, and/or to followthe relative development and health of one or other of such (mixed-sex)twin pregnancies. The present invention may also be utilised in genderdetermination of twin pregnancies, by consideration of the relativevalues for foetal cfDNA compared to counts of Y-chromosome sequencesdetermined from cfDNA (eg by using parallel sequencing approaches). Inthese regards, it should be noted that approaches that usemassively-parallel sequencing of random cfDNA in maternal bloodtypically always count a very low frequency of “Y-chomomosone” sequences(such as between about 0.003% and 0.004% of all sequences, or betweenabout 0.0015% and 0.01% or 0.002% and 0.005% of all sequences) in allfemale pregnancies due to homology of certain Y-chromosome shortsequences to other chromosomes. A cut off of “Y-chromosome” sequencecounts of about 0.005%, or between about 0.003%, 0.004%, 0.006% or0.007%, may therefore be employed for female samples.

As described elsewhere herein, there is also increasing evidence thatthe presence and amount of methylated DNA at certain DMRs is indicativeor prognostic of certain medical conditions that are not associated withpregnancy. Accordingly, in another particular embodiment of the presentinvention, said species of DNA originates from a cell type associatedwith such a medical condition, particularly in those embodiments wheresaid species of DNA is circulating cell-free DNA and said sample is ablood fraction such as plasma or serum. For example, the medicalcondition may be a cell proliferative disorder, such as a tumour orcancer. In particular embodiments, the medical condition is a tumour ora cancer of an organ selected from the list consisting of: liver, lung,breast, colon, oesophagus, prostate, ovary, cervix, uterus, testis,brain, bone marrow and blood; and/or said species of DNA may originatefrom cells of a tumour; particularly where such tumour is a carcinoma orcancer of an organ selected from the group consisting of: liver, lung,breast, colon, oesophagus, prostate, ovary, cervix, uterus, testis,brain, bone marrow and blood.

When used in the context of a medical condition being a tumour orcancer, the present invention includes embodiment wherein said DMR(s)comprises at least one, preferably at least two, methylation site(s)specific for said reagent, and at least one of said DMR is located in aportion of the genome and/or a gene (in particular, when such genomeand/or gene is a human genome or gene) selected from the groupconsisting of: a tumour suppressor gene, p16, SEPT9, RASSF1A, GSTP1.DAPK, ESR1, APC, HSD17B4 and H1C1. In particular, one of said DMR(s), ortwo or more DMRs, may be located in RASSF1A (eg SEQ ID NO. 13) such aslocated between about positions 4,700 bp and 5,600 bp of such RASSF1A;and/or said other region is located between about positions 14,220 bpand 13,350 bp of such RASSF1A. Other particular locations of the DMR(s)and/or other region(s) within RASSF1A for use in this embodiment aredisclosed elsewhere herein. Furthermore, the person of ordinary skillwill now recognise that other DMRs and/or other regions located in otherportions of the genome of in other genes may be identified from therelevant scientific literature (eg, for review, see Elshimali 2013). Inparticular when used in the context of a medical condition being atumour or cancer, the present invention includes embodiments where atleast one or more (additional) other region(s) (preferably two or more)are located in a (eg human) housekeeping gene (such as GAPDH,beta-actin, ALB, APOE or RNASEP). Alternatively for such context, said(additional) other region(s) may be located in the same portion of thegenome and/or gene that feature one or more DMRs (such as those p16,SEPT9, RASSF1A, GSTP1. DAPK, ESR1, APC, HSD17B4 and H1C1).

In yet another particular embodiment of the present invention, saidspecies of DNA originates from a cell type associated with a medicalcondition selected from the group consisting of: an infection/infectiousdisease, a wasting disorder, a degenerative disorder, an (auto)immunedisorder, kidney disease, liver disease, inflammatory disease, acutetoxicity, chronic toxicity, myocardial infarction, and a combination ofany of the forgoing (such as sepsis) and/or with a cell proliferativedisorder, particularly in those embodiments where said species of DNA iscirculating cell-free DNA and said sample is a blood fraction such asplasma or serum. For example, the medical condition may be aninfection/infectious disease, such as one caused by a bacterial, viralor protozoan pathogen, including a pathogen selected from the groupconsisting of: a retrovirus (such as HIV), a herpes virus (such as HSV,EBV, CMV, HHV or VSV), dengue virus, mycobacteria (eg Mycobacteriumtuberculosis), and hantavirus. In certain embodiments, the medicalcondition is sepsis and/or excludes kidney disease.

In all aspects of the present invention, there exist embodiments whereinthe sample is a tissue sample or a sample of biological fluid. Inparticular, the sample is whole blood or a blood fraction (eg, such asplasma or serum). In alterative embodiments, the sample is biologicalfluid selected from the group consisting of: urine, saliva, sweat,ejaculate, tears, phlegm, vaginal secretion, vaginal wash and colonicwash. In more particular embodiments, the sample is a plasma or serumsample from the individual, or is urine from the individual. In otherembodiments, the sample is largely (or essentially) free from cells,and/or is not a whole blood and/or ejaculate sample. In certainembodiments, the sample is not ejaculate if the individual is female andthe sample is not a vaginal wash if the individual is male.

In all aspects of the present invention, the reagent that differentiallymodifies methylated and non-methylated DNA may comprise bisulphiteand/or an agent that selectively digests unmethylated over methylatedDNA (for example, such agent may digest unmethylated DNA but notmethylated DNA). In particular embodiments, the reagent agent comprises:at least one methylation sensitive enzyme; at least one methylationsensitive restriction enzyme; and/or an agent selected from the groupconsisting of: AatII, AciI, AcII, AfeI, AgeI, AgeI-HF, AscI, AsiSI,AvaI, BceAI, BmgBI, BsaAI, BsaHI, BsiEI. BsiWI, BsmBI, BspDI, BsrFI,BssHII, BstBI, BstUI, ClaI, EagI, FauI, FseI, FspI, HaeII, HgaI, HhaI,HinP1I, HpaII, Hpy99I, HpyCH4IV, KasI, MluI, NaeI, NarI, NgoMIV, NotI,NotI-HF, NruI, Nt.BsmAI, Nt.CviPII, PaeR7I, PluTI, PmlI, PvuI, PvuI-HF,RsrII, SacII, SalI, SalI-HF, SfoI, SgrAI, SmaI, SnaBI, TspMI and ZraI.In particular embodiments, said reagent is one selected from the groupconsisting of: BstUI, HhaI and HpaII.

In related embodiments, the reagent may comprise two or more of any ofthe reagents disclosed herein. For example, it may comprise two, three,four, five or more (eg up to seven, eight or ten) methylation sensitiverestriction enzymes, including a reagent comprising or essentiallyconsisting of two or three of the methylation sensitive restrictionenzymes selected from the group consisting of: BstUI, HhaI and HpaII

The use of bisulphite or methylation-sensitive restriction enzymes tostudy differential methylation will be well known to the person ofordinary skill, who may apply teachings of standard texts or adaptationof published methods such as Poon et al (2002), Nygren et al (2010) orYegnasubramanian; et al (2006, Nuc Acid Res 34:e19). By way ofillustration, the inventors provide examples herein that employ the useof methylation-sensitive restriction enzymes as the reagent thatdifferentially modifies methylated and non-methylated DNA. For furtherillustration using bisulphite as reagent, it will be apparent to theperson of ordinary skill that bisulphite-modified DNA methylation sitesmay be detected using eg methylation-specific PCR (such as using primersand/or probes that selectively bind to the bisulphite-modifiedsequences) and/or by the subsequent use of restriction enzymes therecognition site of which is created upon such bisulphite-modification.Methylation-specific PCR (“MSP”) is described by Herman et al(U56200756, EP0954608 and related family members); and a furtherdevelopment of MSP using probe-based PCR (known as “MethylLight”) isdescribed by Laird et al (U56331393, EP1185695 and related familymembers).

In particular embodiments of all aspects of the invention, aquantitative amount of said species of DNA (and/or or said total DNA) isto be detected and/or determined. Accordingly in such embodiments, oneor more (eg each) of said detection steps comprises quantitativedetection and said detected amount of said species of DNA is expressedas a relative concentration of said species of DNA to the total DNApresent in said sample.

If an absolute amount of total DNA is known, then correspondingly anabsolute amount (for example, as represented by a concentration such asμg/mL or genome-equivalents such as Eg/mL) of the species of DNA can bedetermined from such relative concentration. An absolute amount of totalDNA for a sample may be determined, for certain embodiments, byincluding the further steps of: detecting an amount of total DNA in astandard sample of DNA of known amount using the same other regions(s)as used in step (c); and comparing the signal detected from saidstandard sample of DNA to the signal detected in step (c). Such astandard sample of DNA (of known amount/concentration) is readilyavailable from commercial sources, and especially if prepared andanalysed using a dilution series, can readily and efficiently be used todetermine (by interpolation/estimation from the standard curve) anabsolute amount of total DNA present in the sample. Practically, suchstandard curve may be prepared and analysed essentially as described forthe other regions (but in a separate set of standard vessels/reactions),preferably in the same run as the detection of the DMRs/other region(s);and may even use the same reaction master-mix. Accordingly, while the“DMR(s)” of the DNA control may be detected for such standard DNA, sucha signal is not required to generate a standard curve. Accordingly, ifthe signal from a such a standard DNA sample is used to compare, the incertain embodiments where each of said detection steps comprisesquantitative detection, said detected amount of said species of DNA canbe expressed as an absolute amount of said species of DNA in saidsample.

A determined quantitative amount of said species of DNA has utility inassessing the risk of the individual to certain medial conditions and/orif there is sufficient of such species of DNA in the sample to enablefurther analysis of such species of DNA to be conducted efficiently,accurately and/or in a cost effective manner.

Accordingly, certain embodiments of the present invention furtherinclude the step of: comparing the amount of said species of DNAdetected with a threshold amount and/or reference distribution ofamounts, wherein an increase in the (or outlying) amount of said speciesof DNA indicates an increased risk of the individual suffering from ordeveloping a medical condition. Threshold amounts and/or a set ofamounts to form a reference distribution may be obtained from publishedliterature and or empirical studies. For example, using publishedthreshold values (Papantoniou et al 2013, Prenat Diag 33:682) if thetotal cfDNA exceeds an amount of about 7,500 Eg/mL plasma or if thefoetal cfDNA fraction exceeds an amount of about 500 Eg/mL plasma, thenthe woman may be determined to have such an increased risk. Such a riskmay instead or additional be assessed by considering: (i) thefold-increase (eg 1.5, 3, 3.5 or 4-fold increase) of foetal cfDNA(determined for such woman compared to a threshold amount), factoringinto the determination that for later-term pregnancies a higherfold-increase in foetal cfDNA may be utilised (Zeybek et al 2013, JObstet Gynaecol Res 39:632); and/or (ii) into which percentile theamount of cfDNA determined from the woman falls, from consideration of areference distribution of amounts such as those determined from low-riskwomen or those which did not suffer from or develop preeclampsia, forexample if the foetal cfDNA fraction falls within the 90^(th) percentileof such a distribution, then the woman may be considered to have anincreased risk of suffering mild or severe preeclampsia (Jakobsen et al2013, Transfusion 53:1956). Other relevant factors may be considered indetermining a suitable threshold amount. For example, a pregnant womenwho is also suffering from breast cancer, may have a higher bias ofmethylation at RASSF1A present in her plasma due to both factors.

Analogously, certain embodiments of the present invention furtherinclude the step of: comparing the amount of said species of DNAdetected with a threshold amount and/or reference distribution ofamounts, wherein an amount of said species of DNA in excess to saidthreshold (or is not an outlier compared to said population) indicatesthat a diagnosis for an abnormality in the said species of DNA presentin said sample may be performed on, preferably a separate aliquot of DNAof, said sample. For example, if foetal cfDNA fraction is greater thanabout 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.5% of total cfDNApresent in maternal plasma, then there would be sufficient fraction orfoetal cfDNA to effectively conduct a subsequent test to investigate oneor more characteristics of the foetal cfDNA, for example to investigatethe chance or existence of a chromosomal anomaly of mutation comprisedwithin such foetal cfDNA (such as using NIPT based on massively parallelsequencing). In the case of twin pregnancies, the inventors determinethat a minimum foetal fraction of cfDNA for NIPT of a twin pregnancycould be considered to be 8%, or about 5%, 6%, 7%, 9% or 10%, and formonochorionic twin pregnancies with concordant genotypes (apart fromrare exceptions, Chen et al, 2013, Am J Med Genet A, 161A:1817), afoetal cfDNA fraction of 4%, or about 2%, 3% or 5%, would be sufficient.In certain embodiments, the threshold amount(s) may be established by astandard control; for example, established experimentally from a knownsample (or a plurality of known samples) once or separately, and/or athreshold amount(s) that is established (eg a from a sample or pluralityof known samples) at about the same time as the test sample (or testsamples), such as in the same run, in particularly by establishing thethreshold amount(s) by practicing a method of the present invention onsamples contained in wells of a microtitre plates where one or moreknown samples placed in one or more (separate) wells and one or moretest samples placed in other wells. In other embodiments of the presentinvention, a comparison with a threshold amount and/or referencedistribution of amounts is made from the relative amount (such as theratio of) the amount of a first species of DNA to the amount of secondspecies of DNA. For example, an amount of a first species of DNAoriginating from a normal diploid set of human chromosomes 21 would beexpect to show about a 2:2 (ie 1:1) ratio to the amount of a secondspecies of DNA originating from a reference (diploid) set of egchromosome 2. However, in the event of trisomy 21, such a ratio would beexpected to be about 3:2. As will now be understood by the person ofordinary skill, other chromosomal (or partial chromosomal) aneuploidieswould be expect to show other ratios, for example 1:2 in the case of aloss of a complete chromosome, or a partial loss such as a partialdeletion of the location of the first species of DNA, compared to thereference chromosome comprising the location of the second species ofDNA. In certain embodiments, eg if not differentiated such as bymethylation differences, the presence of other DNA (ie in a mixture withsuch other DNA) such as euploid maternal cfDNA in admixture withaneuploid foetal cfDNA could result in modified such ratios depending onthe relative amounts of (euploid) maternal and (aneuploid) foetalcfDNAs. As will also be understood by the person or ordinary skill,modified such ratios may result from other factors such as the relativereaction (eg PCR reaction) efficiency of each amplicon. Accordingly, incertain of such embodiments, the threshold amount is a ratio that is(detectably and/or significantly) greater or smaller than 2:2 (100%)such as about 3:2 (150%), about 2:3 (66%), about 1:2 (50%) or about 2:1(200%); including threshold amounts and/or ratios that are greater thanabout 200%, less than about 50%, or is one selected from the listconsisting of about: 190%, 180%, 170%, 160%, 150%, 140%, 130%, 120%,110%, 105%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, and 55%.Alternatively, in particular embodiments, the threshold amount may bedetermined merely by there being no detectable amount of the first (orsecond) species of DNA, such as in Turners syndrome (a human female witha “45, X” karyotype rather than the fully euploid “46, XX” karyotype).

Comparing and detecting differences between sample distributions andreference distributions, or sample outliers from reference distributionswill be known to the person of ordinary skill, and include the use ofparametric and non-parametric statistical testing such as the use of(one- or two-tailed) t-tests, Mann-Whitney Rank Sum test and others,including the use of a z-score, such as a Median Absolute Deviationbased z-score (eg, such as used by Stumm et al 2014, Prenat Diagn34:185). When comparing a distribution to (or outliers from) a referencedistribution, then in certain embodiments of the invention, thecomparison is distinguished (and/or identified as being significantlydifferent) if the separation of the means, medians or individual samplesare greater than about 1.5, 1.6, 1.7, 1.8, 1.9, 1.95, 1.97, 2.0, orgreater than about 2.0 standard distributions (“SD”) of the referencedistribution; and/or if an individual sample separates from thereference distribution with a z-score of greater than about 1.5, 1.6,1.7, 1.8, 1.9, 2.0, 2.5, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5,3.75, 4.0, 4.5, 5.0 or greater than about 5.0.

In certain embodiments, a parameter (such as a mean, median, standarddeviation, median absolute deviation or z-score) is calculated inrespect of a set of samples within each run, plate or detection/analysisdata-set. In certain of such embodiments, such a calculated parameter isused to identify outliers (such as trisomy samples) from those testsamples detected/analysed in such run, plate or data-set (eg, a“run-specific” analysis). In particular embodiments, such a parameter iscalculated from all test samples without knowledge of the identity ofany outliers (eg a “masked” analysis). In other particular embodiments,such a parameter is calculated from a set of reference samples know tobe (non-outlying) standards (such as samples known to contain cfDNA fromeuploid foetuses) or test samples that are presumed to be (or areunlikely to be) such standards.

In certain embodiments, in the context of a data set a z-score (or anequivalent statistic based on the distribution pattern of replicates ofa parameter) may be calculated to identify an outlying data point(s)(for example, representing an excessive amount of the species of DNAsuch as in the context of a test seeking to identify a pregnant femalepredicted to have or having an increased risk of suffering or developingpreeclampsia, or representing an excessive amount of one chromosome overa reference chromosome such as in the context of a test seeking toidentify a foetus suffering from a chromosomal aneuploidy), the datarepresenting such data point removed from the data set and a subsequentz-score analysis be conducted on the data set to seek to identifyfurther outliers. Such an iterative z-score analysis may be particularhelpful in detection of foetal chromosomal aneuploidies using a methodof the present invention, where sometimes two or more aneuploidy samplesin one run may skew a single z-score analysis, and/or where follow-uptests are available to confirm false positives and hence avoiding falsenegatives is potentially more important that the (initial)identification of false positives.

Accordingly, one other aspect of the present invention relates to amethod to identify at least one sample as an outlier from a set ofsamples each from an individual, said method comprising the steps: (i)calculating an absolute or relative amount of said species or DNA ineach sample of said set using a method of the present invention (such asthe first, second or further aspects); (ii) calculating a mean (ormedian) and a standard deviation (or a median absolute deviation forsaid amount in respect of all samples in the set; (iii) conducting az-score analysis on all samples in the set; (iv) identifying any sampleswith a z-score of greater than about 1.7; (v) repeating steps (ii) to(iv) at least one more time, each time removing from the calculations ofstep (ii) any data in respect of additional samples with a z-score ofgreater than about 1.70. In certain embodiments of such aspect, thez-score is greater than about 1.75, 1.80. 1.85, 1.90, 1.95, 2.00, 2.05,2.10, 2.15, 2.20, 2.25, 2.30, 2.35, 2.40, 2.45 or 2.50; and/or steps(ii) to (iv) are repeated two, three, four, five or between five andabout eight more times. In particular embodiments, steps (ii) to (iv)are repeated until no further samples are identified as outliers. In oneembodiment of such aspect, the z-score cut-off is raised (such as byabout 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45 or 0.50) ineach repeated set of steps. In certain embodiments of such aspect, theamount of said species of DNA is a relative amount of total foetal cfDNAand the outlying sample(s) represent a pregnant female with an increasedrisk of developing a medical condition such as preeclampsia, pretermlabour, intrauterine growth retardation or vanishing twin (inparticular, preeclampsia). In certain embodiments of such aspect, theamount of said species of DNA is a relative amount of a first species offoetal cfDNA located on a chromosome relevant to the chromosomalaneuploidy or within a section of a chromosome relevant to thechromosomal aneuploidy to a second species of foetal cfDNA located on areference chromosome, and the outlying sample(s) represent a pregnantfemale with an increased risk of carrying a aneuploid foetus (such as afoetus with trisomy 21, trisomy 18 or trisomy 13). As will now beappreciated by the person of ordinary skill, an corresponding suchaspect would include where an outlier is identified if the z-score isless than a negative cut-off, such as less that about −1.7. A relatedother aspect of the present invention relates to a computer programproduct comprising a computer readable medium encoded with a pluralityof instructions for controlling a computing system to perform and/ormanage an operation for conducting at least steps (ii) to (v) of thepreceding aspects. In certain embodiments, such program product furthercomprises instructions for controlling a computing system to performand/or manage an operation for conducting step (i).

Therefore, the present invention also includes embodiments wherecomprising a further step of: performing on, preferably with a separatealiquot of DNA of, said sample an in-vitro diagnosis for an abnormalityin said species of DNA present in said sample; preferably wherein, saidspecies of DNA originates from cells of a foetus and/or the placenta ofa foetus, said sample is from a pregnant female and said diagnosis is aprenatal diagnosis. Such diagnosis directed at said species of DNApresent may comprise a step that uses a detection technology selectedfrom the group consisting of: DNA sequencing, SNP analysis, digital PCRand hybridization, and in particular embodiments said detectiontechnology is massively parallel sequencing of DNA, such as massivelyparallel sequencing of random and/or (exon) enriched DNA. In certainembodiments of the present invention, such an in-vitro diagnosis isconducted on the same aliquot of DNA. For example, a method of thepresent invention may be conducted (or modified to so conduct) so as toenable such a diagnosis from the practice of such a method without theneed for a further detection technology. Such methods provideparticularly advantageous solutions to solve the need or more rapid,simpler and less expensive approaches to provide such diagnostic tests.Particular aspects and/or embodiments of the present invention thatprovide for such advantageous solutions are described herein.

Such a diagnosis or test may be directed at the foetal DNA to identify agenetic mutation or chromosomal abnormality of the foetal DNA.Accordingly in certain embodiments, said species of DNA originates fromcells of a foetus and/or the placenta of a foetus, said sample is from apregnant female and said abnormality is a genetic mutation or achromosomal abnormality, such as a chromosomal trisomy, associated witha foetal abnormality and/or a congenital disorder, In particular suchembodiments, the genetic mutation is selected from the group consistingof: colour blindness, cystic fibrosis, hemochromatosis, haemophilia,phenylketonuria, polycystic kidney disease, sickle-cell and disease,Tay-Sachs disease; and/or the chromosomal abnormality is selected fromthe group consisting of: a trisomy (such as trisomy 21, trisomy 18, ortrisomy 13), a sex-chromosome abnormality (such as Turners syndrome,Klinefelter syndrome, [Noonan syndrome,] Triple X syndrome, XXYsyndrome, or Fragile X syndrome or XYY syndrome or XXYY syndrome), achromosomal deletion (such as Prader-Willi syndrome, Cris-du-chatsyndrome, Wolf-Hirschhorn syndrome, or 22q11 deletion syndrome, Duchenemuscular dystrophy), Beckwith-Wiedemann syndrome, Canvan syndrome, andneurofibromatosis. In other embodiments, the genetic mutation orchromosomal abnormality may be one or more selected from those having aclinical utility gene cards (CUGCs) of the EuroGentest2 initiative(www.eurogentest.org). In particular embodiments, the chromosomalabnormality is a trisomy (such as trisomy 21, trisomy 18, or trisomy13), a sex-chromosome abnormality or a chromosomal deletion.

Such diagnosis or test may be directed at a species DNA to identify agenetic mutation or chromosomal abnormality of such DNA that is derivedfrom a cell or cell-type associated with a medical condition.Accordingly in one of such embodiments, said species of DNA originatesfrom cells of a tumour and said abnormality is a genetic mutation or achromosomal abnormality associated with the diagnosis, prognosis orpredictive treatment of a carcinoma or cancer. In particular suchembodiments, the genetic mutation is selected from the group consistingof: a mutation in a tumour suppressor gene (such as TP53 (p53), BRCA1,BRCA2, APC or RB1), a mutation in a proto-oncogene (such as RAS, WNT,MYC, ERK, or TRK) and a DNA repair gene (such as HMGA1, HMGA2, MGMT orPMS2); and/or the chromosomal abnormality is a translocation (such ast(9;22)(q34;q11) [ie, Philadelphia chromosome or BCL-ABL],t(8;14)(q24;q32), t(11;14)(q13;q32), t(14;18)(q32;q21),t(10;(various))(q11;(various)), t(2;3)(q13;p25), t(8;21)(q22;q22),t(15;17)(q22;q21), t(12;15)(p13;q25), t(9;12)(p24;p13),t(12;21)(p12;q22), t(11;18)(q21;q21), t(2;5)(p23;q35),t(11;22)(q24;q11.2-12), t(17;22), t(1;12)(q21;p13),t(X;18)(p11.2;q11.2), t(1;19)(q10;p10), t(7,16)(q32-34;p11),t(11,16)(p11;p11), t(8,22)(q24;q11) or t(2;8)(p11;q24)).

In particular embodiments of the present invention:

-   -   the presence of methylated DNA at a first DMR (or a first set of        two or more DMRs), for example detected in a detection step (b)        of a method of the present invention, is used to indicate the        presence of an amount of a first species of DNA in said sample        and the absence of methylated DNA at said first DMR (or first        set of DMRs) indicates the absence of said first species of DNA        in said sample; preferably wherein, said first species of DNA        originates from cells of a foetus and/or the placenta of a        foetus (for example, from a chromosome relevant to a chromosomal        aneuploidy such as human chromosome 21), said sample is from a        pregnant female and at least one of said first DMR (first set of        DMRs) is one as set forth herein; and    -   the presence of methylated DNA at a second DMR (or second set of        two or more DMRs)), for example detected in the same or        different a detection step (b) of a method of the present        invention, is used to indicate the presence of an amount of a        second species of DNA in said sample and the absence of        methylated DNA at said second DMR (or second set of DMRs)        indicates the absence of said second species of DNA in said        sample; preferably wherein, said second species of DNA        originates from cells of a foetus and/or the placenta of a        foetus, said sample is from a pregnant female and at least one        of said second DMR (or second set of DMRs) DMR is one set forth        herein; and    -   a first amount of total DNA present in said sample is detected,        for example in a detection step (c) of a method of the present        invention, using a first region that is not differently        methylated between said first species of DNA and the DNA not        originating from cells of said type, the modification of which        first other region by said reagent is insensitive to methylation        of DNA, wherein said first other region is located between about        20 bp and about 20 kb upstream or downstream of said first DMR        (or at least one of first set of DMRs); and    -   a second amount of total DNA present in said sample is detected,        for example in the same or different a detection step (c) of a        method of the present invention using a second region that is        not differently methylated between said second species of DNA        and the DNA not originating from cells of said type, the        modification of which second other region by said reagent is        insensitive to methylation of DNA, wherein said second other        region is located between about 20 bp and about 20 kb upstream        or downstream of said second DMR (or at least one of said second        set of DMRs).

By way of graphical description, a schematic representation of thegeneral arrangement of the DMR(s), the other region(s) and thedetectable label(s), as used for such an embodiment of the first aspectof the present invention, is presented in FIG. 6. (1) The presence ofmethylation in a first species of DNA (such as a particular chromosomefor example human chromosome 21) at DMR1 is detected in the context ofan other region (“OR1”) which is located within the same portion of thegenome (eg, between about 20 bp and about 20 kb upstream or downstreamof) DMR1. (2) Optionally, additional DMRs and/or ORs (such as DMR2and/or OR2, and up to DMRn and ORn) may be detected, and pairs of suchadditional DMRs and ORs may each be co-located in a same portion of thegenome (eg, between about 20 bp and about 20 kb upstream or downstreamof) as each other, and in each case all detecting the same first speciesof DNA (such as the same chromosome for example human chromosome 21).Optionally, (3) the presence of methylation in such first species of DNAis detected at multiple DMRs, using the same detectable label(s) and/or(4) the first amount of total DNA detected using at least one OR (OR1,and optionally, OR2 or up to ORn) is detected using different detectablelabel(s) to those used to detect methylation at the DMR(s) representingthe first species of DNA (optionally, the detectable label(s) used isthe same for all the ORs). (1′) In such an embodiment, the first speciesof DNA is detected, identified and/or quantified in comparison to thatof a second species of DNA (such as a reference chromosome for examplehuman chromosome 2). Such second species of DNA is detected as describedfor first species of DNA, but using different DMR(s) andOR(s)—represented by the “prime” after each on the figure, and withreference to the explanatory labels. In those of such embodiments wherethe detection of DMR(s), OR(s), DMR(s)′ and OR(s)′ are made using thesame aliquot of DNA of said sample, in the same reaction/detectionvessel, and/or effectively simultaneously with each other (for exampleby multiplex real-time quantitative probe-based PCR as described herein,such as by using at least one labelled probe specific for each of saidDMR(s) and other regions(s)), then such embodiments include those wherethe detectable label(s) (3), (4), (3)′ and (4)′ are different, and/orcan be separately detected and/or quantified.

By way of graphical description, a schematic representation of thegeneral arrangement of the DMRs, the other region(s) and the detectablelabel(s), as used for such an embodiment of the second aspect of thepresent invention, is presented in FIG. 7. (1) The presence ofmethylation in a first species of DNA (such as a particular chromosomefor example human chromosome 21) at two or more DMRs, DMR1 and DMR2(and, optionally, up to DMRn), is in each case detected using the samedetectable label(s). (2) Optionally, an other region (“OR”) which islocated within a same portion of the genome (eg, between about 20 bp andabout 20 kb upstream or downstream of) one of the DMRs. (3) The firstamount of total DNA detected using at least one OR (OR1, and optionally,OR2 or up to ORn) is detected using different detectable label(s) tothose used to detect methylation at the DMRs (optionally, the detectablelabel(s) used is the same for all the ORs). (4) Optionally, methylationat more than two DMRs is so detected, and/or the first amount of totalDNA is detected at more than one OR. (1′) In such an embodiment, thefirst species of DNA is detected, identified and/or quantified incomparison to that of a second species of DNA (such as a referencechromosome for example human chromosome 2). Such second species of DNAis detected as described for first species of DNA, but using differentDMRs and, optionally, ORs—represented by the “prime” after each on thefigure, and with reference to the explanatory labels. In those of suchembodiments where the detection of DMR(s), OR(s), DMR(s)′ and OR(s)′ aremade using the same aliquot of DNA of said sample, in the samereaction/detection vessel, and/or effectively simultaneously with eachother (for example by multiplex real-time quantitative probe-based PCRas described herein, such as by using at least one labelled probespecific for each of said DMR(s) and other regions(s)), then suchembodiments include those where the detectable label(s) (3), (4), (3)′and (4)′ are different, and/or can be separately detected and/orquantified.

The practice of such a method as described in the previous paragraphs,can enable the relative detection (or amount) of the first species ofDNA (for example, from a chromosome, or part thereof, related to achromosomal aneuploidy such as human chromosome 21) and the secondspecies of DNA (for example, from a reference chromosome, or partthereof, such as chromosome 2), and hence aid the rapid, simple andcost-effective detection, identification or diagnosis of chromosomalabnormalities, such as a chromosomal aneuploidy in a foetus. Such anapproach may be more easily established and practiced in manylaboratories, requiring for example, a relatively simple, reliable andcost-effective quantitative PCR machine; and not requiring expensive andspecialised high-throughput next-generation sequencing machines. Indeed,in certain of such embodiments of the present invention, detection instep (b) of said first and second DMR (or set of DMRs) and saiddetection in step (c) of said first and second other regions are madeusing the same aliquot of DNA of said sample, and in the samereaction/detection vessel, and effectively simultaneously for such DMRsand other regions, and using: (x) a different detectable label(s) foreach of said first and second DMR (or set of DMRs); and (y) furtherdifferent detectable label(s) for each of said first and second otherregions. The relative detection, identification or quantification of thefirst and the second species of DNA (via the first (set of) DMR(s) andthe second (set of) DMR(s)) is, in those embodiments conducted in thesame reaction/detection vessel and effectively simultaneously,advantageously made by the use of detectable labels that can distinguishthe first from the second (set of) DMRs and their corresponding firstand second other regions. In a particular further such embodiment wheredetection (b) and (c) is conducted in the same reaction/detection vesseland effectively simultaneously includes where said detection in step (b)and said detection in step (c) are made by multiplex real-timequantitative probe-based PCR using at least one labelled probe specificfor each of said DMRs and other regions.

In all embodiments of the present invention, particularly thosedetecting a plurality (such as two or more) species of DNA in thesample, the agent may comprise at least one methylation sensitiverestriction enzyme, such as one or more of those described elsewhereherein. Alternatively, or additionally, in such embodiments the agentmay comprise bisulphite, particular for those methods that utilise MSPor MethylLight detection approaches as described herein.

In all embodiments of the present invention, particularly thosedetecting a plurality (such as two or more) species of DNA in thesample, one or more (preferably each) of said detection steps comprisesquantitative detection. For example, in certain embodiments, saiddetected amount of each of said species of DNA is expressed as arelative concentration of said species of DNA to the respective amountof total DNA detected in said sample from the respective other region.In further of such embodiments, the methods of the present invention canfurther comprise the steps of detecting an amount of total DNA in astandard sample of DNA of known amount using the same other regions asused in step (c); and comparing the signal detected from said standardsample of DNA to the respective signal detected in step (c) for each ofthe other regions. In alterative certain embodiments, one or more(preferably each) of said detection steps comprises quantitativedetection and said detected amount of said species of DNA is expressedas an absolute amount of each or said species of DNA in said sample.

In those embodiments of the present invention that quantitatively detecta plurality (such as two or more) species of DNA in the sample, suchmethods can further comprise the steps of:

-   -   determining the relative amount, preferably a ratio, of: (x)        said first species of DNA detected with the first DMR (or first        set of two or more DMR); and (y) said second species of DNA        detected with the second DMR (or first set of two or more DMRs);        and    -   comparing said relative amount or ratio with a threshold and/or        reference distribution of amount(s) or ratio(s), wherein: a        relative amount or ratio that is higher or lower than said        threshold and/or reference distribution of amount(s) or ratio(s)        indicates the presence of an abnormality in said first and/or        second species of DNA present in said sample.

Such embodiments are particularly preferred when the presence of theabnormality to be indicated by the method is a chromosomal abnormalitysuch as a chromosomal abnormality is associated with a foetalabnormality and/or congenital disorder. For example, such a chromosomalabnormality may be selected from the group consisting of: a trisomy(such as trisomy 21, trisomy 18, or trisomy 13), a sex-chromosomeabnormality (such as Turners syndrome, Klinefelter syndrome, [Noonansyndrome,] Triple X syndrome, XXY syndrome, or Fragile X syndrome or XYYsyndrome or XXYY syndrome), a chromosomal deletion (such as Prader-Willisyndrome, Cris-du-chat syndrome, Wolf-Hirschhorn syndrome, or 22q11deletion syndrome, Duchene muscular dystrophy), Beckwith-Wiedemannsyndrome, Canvan syndrome, and neurofibromatosis. Of most relevance, interms of prevalence and hence medical and social significance is wherethe chromosomal abnormality is a trisomy, such as one selected from thelist consisting of trisomy 21, trisomy 18, or trisomy 13.

One further aspect of the present invention relates to a method fordetecting a chromosomal aneuploidy in a foetus carried by a pregnantfemale, said method comprising the steps:

-   (A) Determining, using a method of the present invention (such as a    method of the first and/or second aspect of the invention) in any of    the embodiments described herein (or others), in a sample taken from    said pregnant female the amount of a first species of DNA that    originates from cells of a foetus and/or the placenta of a foetus,    wherein said first species of DNA is located on a chromosome    relevant to the chromosomal aneuploidy or within a section of a    chromosome relevant to the chromosomal aneuploidy, and wherein said    first species of DNA that originates from cells of a foetus and/or    the placenta of a foetus is distinguished from its counterpart of    maternal origin in the sample due to differential DNA methylation;-   (B) Determining, using a method of the present invention (such as a    method of the first and/or second aspect of the invention) in any of    the embodiments described herein (or others), the amount of a second    species of DNA that originates from cells of a foetus and/or the    placenta of a foetus in said sample, wherein said second species of    DNA is located on a reference chromosome, and wherein said second    species of DNA that originates from cells of a foetus and/or the    placenta of a foetus is distinguished from its counterpart of    maternal origin in the sample due to differential DNA methylation;-   (C) determining the relative amount, preferable the ratio, of the    amounts from (A) and (B); and-   (D) comparing said relative amount or ratio with a threshold and/or    reference distribution of amount(s) or ratio(s), wherein: a relative    amount or ratio that is higher or lower than said, threshold and/or    reference distribution of amount(s) or ratio(s) indicates the    presence of the chromosomal aneuploidy in the foetus.

In such further aspect, said amount of the first species of DNA and saidamount of the second species of DNA is determined using a method of thefirst and/or the second aspect of the invention (or any embodimentsthereof). Accordingly, when using a method of the first aspect of thepresent invention, the amount of the first species of DNA may bedetermined by the use (in step (b) of such method) of one or more DMRsand the use (in step (c) of such method) of at least one OR; wherein theOR is located between about 20 bp and about 20 Kb upstream or downstreamof said DMR; and in this further aspect wherein the other region and theDMR are located on a chromosome relevant to the chromosomal aneuploidyor within a section of a chromosome relevant to the chromosomalaneuploidy. Alternatively, when using a method of the second aspect ofthe present invention, the amount of the first species of DNA may bedetermined by the use (in step (b) of such method) of two or more DMRsand the use (in step (c) of such method) of at least one OR; whereinsaid detection in such step (b) and said detection in such step (c) aremade using the same aliquot of DNA of said sample, and in the samevessel, and effectively simultaneously for such DMRs and otherregion(s), and using: (x) the same detectable labels(s) for each of saidDMRs; and (y) a different detectable label(s) for said other region(s);and where at the least the two DMRs are located on a chromosome relevantto the chromosomal aneuploidy or within a section of a chromosomerelevant to the chromosomal aneuploidy. In respect of each suchalterative, examples of suitable DMRs and other regions that are locatedon such chromosome or chromosomal region are described elsewhere herein,and such examples are specifically encompassed in embodiments of suchfurther aspect. As will be now understood by the person of ordinaryskill, the amount of the second species of DNA may be determined using amethod of the first or second aspect of the invention; analogously tothat for the determination of the amount of the first species of DNA asjust described, except that the DMR(s) (and at least one OR if themethod of the first aspect of the invention used for such determination)are located on a reference chromosome. Hence, also included in thisfurther aspect are embodiments that use DMRs and/or ORs describedelsewhere herein as being located on such a reference chromosome. Theuse of a method of either the first or second aspect of the presentinvention to determine the amount of the first species of DNA in step(A) of this further aspect and/or the amount of the second species ofDNA in step (B) of this further aspect, provides that the amount(s) ofsuch species of DNA to be more suitably determined (eg with moreaccuracy and/or precision) than merely by determination of an amountbased only prior-art methods, such as only on a single DMR.

A schematic representation of the differentially methylated regions(“DMR”) and other regions (“OR”) that may be used for such furtheraspect and based on a method of the first aspect of the presentinvention, is shown by FIG. 6, and as described above. Correspondingly,an alternative schematic representation of the differentially methylatedregions (“DMR”) and other regions (“OR”) that may be used for suchfurther aspect and based on a method of the second aspect of the presentinvention, is shown by FIG. 7, and as described above.

An alternative aspect related to the second aspect of the presentinvention relates to an alternative method for detecting in a samplefrom an individual an amount of a species of DNA originating from cellsof a given type, which sample comprises said species of DNA in admixturewith differentially methylated DNA not originating from cells of saidtype; said method comprising the steps:

-   (a) treating the DNA present in said sample with a reagent that    differentially modifies methylated and non-methylated DNA; and-   (b) detecting in said sample the presence of methylation in said    species of DNA at two or more DMRs that are differently methylated    between said species of DNA and the DNA not originating from cells    of said type the modification of DNA of such DMRs by said reagent is    sensitive to methylation of DNA, wherein the presence of methylated    DNA at one or more of said DMRs indicates the presence of said    amount of species of DNA in said sample and the absence of    methylated DNA at said DMRs indicates the absence of said species of    DNA in said sample,    wherein, said detection in step (b) is made using the same aliquot    of DNA of said sample, and in the same reaction/detection vessel,    and effectively simultaneously for such DMRs, and using (x)    multiplex real-time quantitative PCR; and (y) at least two labelled    probes each of which specific for one of said DMRs and that are    labelled with the same detectable label(s) for each of said DMRs.    Such an alternative method of the present invention is not intended    to be practiced on the human or animal body; for example it is    intended to be practiced in an in-vitro manner. Further    characterisation of any of the features of this alternative method    of the present invention (or any combination of such features) can    include the characterisations (and their combinations) as described    elsewhere herein in respect of the first aspect of the invention. In    particular embodiments of this alternative method of the present    invention, the reagent comprises one or more methylation sensitive    restriction enzyme, such as one (or a combination thereof) as    disclosed herein.

In an additional aspect, the invention relates to a method for detectingan increased risk of an individual suffering from or developing amedical condition, said method comprising the steps:

-   (i) conducting a method of the present invention that determines a    quantitative amount said species of DNA (and/or total DNA) in the    sample; and-   (ii) comparing the amount of said species of DNA detected with a    threshold amount and/or a reference distribution of amounts,    wherein an increase in the (or outlying of) amount of said species    of DNA (and/or total DNA) indicates an increased risk of the    individual suffering from or developing said medical condition.

A further additional aspect of the invention relates to a composition(eg, one that is useful for, or used in, a method of the presentinvention), said inventive composition comprising, either (1):

-   -   one pair of PCR primers for amplifying one of said DMRs as set        forth anywhere herein;    -   one pair of PCR primers for amplifying said other region as set        forth anywhere herein;    -   one labelled probe for quantitative probe-based PCR, which        specific for said DMR; and    -   one labelled probe for quantitative probe-based PCR specific for        said other region and labelled with different detectable        label(s) to the probe used for said DMR; or (2);    -   two pairs of PCR primers, each pair for amplifying one of said        two of more DMRs as set forth anywhere herein;    -   one pair of PCR primers for amplifying said other region as set        forth anywhere herein;    -   two labelled probes for quantitative probe-based PCR, each of        which specific for one of said DMRs, and labelled with the same        detectable labels(s) for each of said probe; and    -   one labelled probe for quantitative probe-based PCR specific for        said other region and labelled with different detectable        label(s) to the probes used for said DMRs.

Such a composition of the present invention may further comprise, either(1):

-   -   a further pair of PCR primers for amplifying a second DMR as set        forth anywhere herein; and a further labelled probe for        quantitative probe-based PCR specific for said DMR and labelled        with detectable label(s), optionally that is the same as that        used for the probe(s) specific the first other region; and/or    -   a further pair of PCR primers for amplifying a second other        region as set forth anywhere herein; and a further labelled        probe for quantitative probe-based PCR specific for said other        region and labelled with detectable label(s) that is different        to those used probes for said DMRs; and optionally that is the        same as that used for the probe(s) specific the first other        region; or (2).    -   a further pair of PCR primers for amplifying a second other        region as set forth anywhere herein; and    -   a further labelled probe for quantitative probe-based PCR        specific for said other region and labelled with detectable        label(s) that is different to those used probes for said DMRs;        and optionally that is the same as that used for the probe(s)        specific the first other region.

A yet further additional aspect of the invention relates to a kit (forexample a kit of separate components; such as a kit of holders orvessels, each holding a different component of the kit), such kitcomprising a set of primers and probes as comprised in a composition ofthe present invention. A kit of the present invention may compriseadditional components. For example, the kit may additionally comprise:(i) a printed manual or computer readable memory comprising instructionsto use said primers and probes, including to use them to practice amethod of the present invention and/or to produce or use a compositionof the present invention; and/or (ii) one or more other item, componentor reagent useful for the practice of a method of the present invention;and/or the production or use of the composition of the presentinvention, including any such item, component or reagent disclosedherein, such as a reagent that differently modifies methylated andnon-methylated DNA as set forth anywhere herein.

In particular embodiments of the composition or the kit, one or more ofthe primers or probes comprised therein comprises (or consists of) aprimer or probe sequence selected from one set forth in TABLE 1 and/orTABLE 8. In certain of such embodiments, the composition or the kitcomprises the pair or primers and a probe as set forth in TABLE 1 foreach of (x) one of the DMRs; and (y) one of the ORs; and in further suchembodiments, the pair or primers and a probe as set forth in TABLE 1 forall of (x) the two the DMR; and (y) the two ORs set forth therein. Inalternative such embodiments, the composition or the kit comprises thepair or primers and a probe as set forth in TABLE 8 for all of (x) thetwo the DMR; and (y) the two ORs set forth therein. In any of suchembodiments, the probes may be labelled with the label/quencher (andoptionally a minor grove binding moiety) as set forth in the respectivetable for such probe.

Another further aspect of the invention relates to a computer programproduct comprising a computer readable medium encoded with a pluralityof instructions for controlling a computing system to perform and/ormanage an operation for determining: (x) an increased risk of anindividual suffering from or developing a medical condition and/or (y)if a diagnosis for an anomaly in a species of DNA originating from cellsof a given type may be performed, in each case from a sample from anindividual comprising a species of DNA originating from cells of a giventype in admixture with differently methylated DNA not originating fromcells of said type, the DNA in present in said sample being treated witha reagent that differentially modifies methylated and non-methylated DNAas set forth herein; said operation comprising the steps of:

-   -   receiving: (i) one signal representing the (essentially        simultaneous) quantitative detection of methylation at one or        more (or two or more) DMRs as set forth in step (b) as described        anywhere herein; and (ii) one signal representing the        (essentially simultaneous) quantitative detection of total DNA        using at least one other region as set forth in step (c) as        described anywhere herein;    -   determining a parameter from the signals (i) and (ii), wherein        the parameter represents a quantitative amount of said species        of DNA (and/or said total DNA);    -   comparing the parameter to with a threshold amount and/or        reference distribution of amounts; and    -   based on such comparison, determining a classification of        whether, respectively, (x) an increased risk of an individual        suffering from or developing a medical condition exists;        and/or (y) a diagnosis for an anomaly in a species of DNA        originating from cells of a given type may be performed.

In certain embodiments, a computer program product of the presentinvention the operation further comprises steps of: receiving a furthersignal representing the quantitative detection of total DNA in astandard sample of DNA as set forth anywhere else herein; and comparingsaid signal with the signal representing the essentially simultaneousquantitative detection of total DNA using at least one other region, soas to determine said parameter that represents an absolute quantitativeamount of said species of DNA.

In particular embodiments, the computer program product of the presentinvention is for an operation for determining if a diagnosis for ananomaly in said species of DNA may be performed, and said operationfurther comprises the step of determining from said parameter a numberof random and/or enriched DNA molecules to be sequenced from, preferablyfrom a separate aliquot of DNA of, said sample as part of saiddiagnosis.

In other particular embodiments, the computer program product of thepresent invention is for an operation that further comprises the stepsof:

-   -   receiving: (i) one signal representing the quantitative        detection of methylation at a second DMR (or set of two or more        DMRs) as set forth in step (b) above; and (ii) one signal        representing the quantitative detection of total DNA using a        second other region as set forth in step (c) above;    -   determining a second parameter from the signals (i) and (ii),        wherein the parameter represents a quantitative amount of said        second species of DNA;    -   determining the relative amount, preferable the ratio, of said        parameter and said second parameter;    -   comparing said relative amount or ratio with a threshold and/or        reference distribution of amount(s) or ratio(s); and    -   based on such comparison, determining a classification of        whether an abnormality in said species of DNA or second species        of DNA present in said sample

Such embodiments of the computer program product are particularlypreferred when the presence of the abnormality to be indicated by themethod is a chromosomal abnormality such as a chromosomal abnormality isassociated with a foetal abnormality and/or congenital disorder. Forexample, such a chromosomal abnormality may be selected from the groupconsisting of: a trisomy (such as trisomy 21, trisomy 18, or trisomy13), a sex-chromosome abnormality (such as Turners syndrome, Klinefeltersyndrome, [Noonan syndrome,] Triple X syndrome, XXY syndrome, or FragileX syndrome or XYY syndrome or XXYY syndrome), a chromosomal deletion(such as Prader-Willi syndrome, Cris-du-chat syndrome, Wolf-Hirschhornsyndrome, or 22q11 deletion syndrome, Duchene muscular dystrophy),Beckwith-Wiedemann syndrome, Canvan syndrome, and neurofibromatosis. Ofmost relevance, in terms of prevalence and hence medical and socialsignificance is where the chromosomal abnormality is a trisomy, such asone selected from the list consisting of trisomy 21, trisomy 18, ortrisomy 13, in particular trisomy 21.

One embodiment of operations performed and/or controlled by the computerprogram product of the invention is depicted in FIG. 5. Operation (A)receives signals (1) and (2) that represent, respectively, themethylation at the DMR(s) and the total DNA, and optionally signal (3)then represents an amount of total DNA from a standard sample. Operation(A) determines a parameter (4) from signals (1), (2) and optional (3)that represents a relative or absolute amount of DNA (eg from foetal vstotal DNA). This parameter (4) is compared by operation (B) against athreshold amount (5) and/or a reference population of amounts (6) so asto classify (7) the risk of an individual suffering from a medialcondition and/or if a diagnosis for an anomaly in either of the DNA inthe sample may be performed.

It is to be understood that application of the teachings of the presentinvention to a specific problem or environment, and the inclusion ofvariations of the present invention or additional features thereto (suchas further aspects and embodiments), will be within the capabilities ofone having ordinary skill in the art in light of the teachings containedherein.

Unless context dictates otherwise, the descriptions and definitions ofthe features set out above are not limited to any particular aspect orembodiment of the invention and apply equally to all aspects andembodiments which are described.

All references, patents, and publications cited herein are herebyincorporated by reference in their entirety.

Certain aspects and embodiments of the invention will now be illustratedby way of example and with reference to the description, figures andtables set out herein. Such examples of the methods, uses and otheraspects of the present invention are representative only, and should notbe taken to limit the scope of the present invention to only suchrepresentative examples.

Example 1: Use of the Method of the Invention in NIPT in MultiplePregnancies, Including in Cases of Vanishing Twins

Sample collection, processing and DNA extraction:

36 blood samples from women pregnant with multiple gestations (mono-,di- and trichorionic twin and triplet pregnancies) were collectedbetween Nov. 6, 2012 and Nov. 16, 2013, for research & development (R&D)purposes and as part of routine non-invasive prenatal testing (NIPT)laboratory procedure. One blood sample came from a woman pregnant withtriplets, the remaining 35 samples came from twin pregnancies. From eachpregnant woman carrying a multiple pregnancy two samples each with 7-10ml venous blood were collected using Streck cell-free DNA bloodcollection tubes (Streck). The blood samples were shipped to thediagnostic laboratory with a maximum delivery time of 4 days. Otherblood samples from pregnant females analysed herein were similarlycollected.

Plasma preparation was performed by centrifugation (1600 g for 10 min at4° C.) and plasma separation followed by a second centrifugation step(16000 g for 10 min at 4° C.). Extraction of total cell-free DNA (cfDNA)was performed with QIAamp Circulating Nucleic Acid Kit (Qiagen)according to the manufacturer protocol using 3.0-4.0 ml plasma with afinal elution volume of 60 ul AVE-buffer (Qiagen).

DNA Quantification:

Foetal cell-free DNA (foetal cfDNA) was detected and quantified inrelation to total cell-free DNA (total cfDNA) in order to determine thefoetal cfDNA fraction as both a relative concentration and absoluteamount using a method of the present invention. From the elutedcell-free DNA, 11 ul were digested with the CpG-methylation sensitiveenzymes HhaI (0.4 U/ul), HpaII (0.3 U/ul) and BstUI (0.3 U/ul) in a 22ul reaction using CutSmart™ Buffer (New England Biolabs). The reactionwas incubated for 60 min at 37° C. and 60 min at 60° C. 10 ul from thedigestion reaction was used as template DNA for quantitative probe-basedPCR (reactions were conducted in duplicate), described briefly asfollows.

A 25 ul PCR reaction using a 2-fold concentrated PCR master mix(QuantiFast Multiplex PCR Kit, Qiagen) was conducted. Primers that spanCpG methylation sensitive restriction enzyme sites of the respectiveregion that is differentially methylated between foetal and maternal DNA(as a DMR) were used in combination with FAM-labelled probes for suchDMRs, and primers that do not span any restriction enzyme sites, another region that is not differentially methylated between foetal andmaternal DNA (as an OR) are used in combination with VIC-labelled probesfor such ORs. The sequences of the primers and labelled probes used inthis example are described in TABLE 1, and the thermocycler profilesused for the quantitative probe-based (TaqMan) PCR (LightCycler 480 IIInstrument; Roche) are described in TABLE 2. In this example, the probesused to detect the presence of the two DMRs, are each labelled with thesame detectable fluorescein amidite (FAM) fluorescent moiety, and eachwith the same minor binding grove (MGB) non-fluorescent quencher (NFQ)moiety, and the probes used to detect the presence of the two ORs, areeach labelled with the same detectable VIC (life Technologies)fluorescent moiety, and each with the same MGBNFQ moiety.

TABLE 1 Quantitative (probe-based) PCR components SEQ ul Final ID Stockfor uM Region Component Sequence (5′-3′)** No.* Conc 1x Conc Master-mixN/A 2x 12.5   1x RASSF1A DMR1-For ATT GAG CTG CGG GAG CTG GC  1 100 uM 0.35   1.4   DMR DMR1-Rev TGC CGT GTG GGG TTG CAC  2 100 uM  0.35  1.4   DMR1-Probe [FAM]-ACC CGG CTG GAG CGT-[MGBNFQ]  3 100 uM  0.035 0.14  RASSF1A OR1-For GGT CAT CCA CCA CCA AGA AC  4 100 uM  0.35  1.4   Other OR1-Rev TGC CCA AGG ATG CTG TCA AG  5 100 uM  0.35   1.4  region OR1-Probe [VIC]-GGG CCT CAA TGA CTT CAC GT-[MGBNFQ]  6 100 uM 0.035  0.14  TBX3 DMR2-For GGT GCG AAC TCC TCT TTG TC  7 100 uM  0.35  1.4   DMR DMR2-Rev TTA ATC ACC CAG CGC ATG GC  8 100 uM  0.35   1.4  DMR2-Probe [FAM]-CCC TCC CGG TGG GTG ATA AA-[MGBNFQ]  9 100 uM  0.035 0.14  TBX3 OR2-For TGT TCA CTG GAG GAC TCA TC 10 100 uM  0.35   1.4  Other OR2-Rev CAG TCC ATG AGG GTG TTT G 11 100 uM  0.35   1.4   regionOR2-Probe [VIC]-GAG GTC CCA TTC TCC TTT-[MGBNFQ] 12 100 uM  0.035  0.14 General DMSO N/A 100%  0.025  0.625 reagents MgCl2 N/A  50 mM  2     1     DNA sample N/A 10     Water — Total 25     *Only nucleotidesequence listed, without dyes/quenchers **The dyes/quenchers used foreach probe are shown in “[]” parentheses

TABLE 2 Thermocycler profiles Step Temperature Time Cycles Analysis modePre-incubation 95° C.  5 min 1 None Denaturation 95° C. 10 sec 45Quantification Annealing 60° C. 10 sec None Elongation 72° C.  8 secSingle Cooling 40° C. None

The assay design used in this example is based on two marker DMRs whichare described to be hypomethylated in maternal DNA and hypermethylatedin foetal DNA (Nygren, et al, 2010: Clin Chem 56, 1627; Chan et al,2006: Clin Chem 42, 2211; Chiu et al, 2007: Am J Pathol 170, 941), andtwo other regions (ORs) not differentially methylated between maternaland foetal DNA which are each located between about 20 bp and 20 kb oftheir DMR. In particular, the methylation insensitive locus located inRASSF1A is located between 8 kb and 9 kb (8.97 kb) downstream of themethylation sensitive locus located in RASSF1A, and the methylationinsensitive locus located in TBX3 is located between 10 kb and 11 kp(10.64 kb) downstream of the methylation sensitive locus located inTBX3. FIG. 2 depicts the respective arrangements and detectionmodalities of the two DMRs and the two other regions used in thisexample.

Parallel probe-based quantitative PCR reactions were performed (inseparate reactions within the same PCR run) using for template a serialdilution of male genomic DNA (Promega) having known concentrations as astandard. The foetal cfDNA fraction was calculated by relativequantification of signals in the FAM channel (DMR; ie detecting foetalcfDNA) versus the VIC channel (ORs; ie detecting total cfDNA), and theabsolute total cfDNA amount was calculated by absolute quantification ofsignals in the VIC channel obtained from the sample compared to the VICchannel obtained from the dilution series of standard DNA of knownconcentration. Such relative and absolute quantifications were conductedusing LightCycler 480 Software release 1.5.0 (Roche).

Maternal Plasma DNA Sequencing and Data Analysis to Identify FoetalAneuploidy:

DNA sequencing libraries were prepared using NEBNext Ultra™ DNA LibraryPrep Kit from Illumina. Libraries were prepared according to themanufacturer protocol automated on a Hamilton STARplus robot. Libraryquality and quantity was measured using a Bioanalyzer instrument(Agilent) and a Qbit Fluorometer (Invitrogen). Based on the libraryquantification dilutions and equimolar pools of 12 samples per pool wereprepared. The pooled samples were sequenced on one lane of an Illuminav3 flow cell on an Illumina HiSeq2000 sequencer. Clonal clusters weregenerated using TruSeq SR Cluster Kit v3-cBot-HS on a cBot Clustergeneration System according to the manufacturer protocol. Bioinformaticanalysis to identify foetal chromosomal aneuploidy was carried out asdescribed previously, with z-scores ≥3 indicating the presence of afoetal trisomy 21 (Stumm et al 2014, Prenat Diag 34:185). In cases of apositive test result for foetal aneuploidy from this method, the resultwas confirmed by invasive diagnostic methods.

Results:

Characteristics, % foetal fraction of cfDNA and aneuploidy test resultsfor the blood samples are given in TABLE 3. There were two positive testresults indicating foetal trisomy 21. Both were confirmed by karyotypingafter amniocentesis; thus, the false positive rate in this study was 0%.One blood sample represented monochorionic twins with concordantkaryotypes [47,XY,+21] and the other one represented dichorionic twinswith discordant karyotypes [47,XY,+21 and 46,XX]. In both samples thefoetal fraction was as high as 18.0 and 24.8%, respectively. All otherNIPT results were negative for trisomies 21, 18 and 13. There is noevidence for false-negative NIPT results so far in the pregnanciesincluded in this study. Nevertheless, a number of pregnancies are stillon-going (with the last birth of the patients expected in mid May 2014)and therefore, the final detection rate is still uncertain.

TABLE 3 Characteristics and NIPT results for the collected blood samplesFoetal DNA Gestational Chr13 Chr18 Chr21 fraction age No. of foetuses,chorinicity Sample z-score z-score z-score (%) (p.m.) amnionicity NIPTresult LCMPC05 1.3 −1.0 −0.8 16.7 11 + 5 3, trichorionic, triamnioticnegative LCMPC06 −0.4 1.1 8.5 18.0 13 + 2 2, monochorionic, n.a. T21positive LCMPC07 −1.0 0.3 0.9 7.9 19 + 0 2, dichorionic, diamnioticnegative LCMPC08 0.7 1.2 0.0 16.5 18 + 1 2, dichorionic, diamnioticnegative LCMPC09 0.6 −0.8 0.7 8.9 11 + 5 2, monochorionic, diamnioticnegative LCMPC10 0.3 0.7 −0.7 17.6 20 + 4 2, dichorionic, diamnioticnegative LCMPC11 −0.9 −0.8 0.7 11.5 23 + 0 2, dichorionic, diamnioticnegative LCMPC12 −0.9 −0.7 −2.0 13.3 11 + 1 2, monochorionic, diamnioticnegative LCMPC13 1.3 0.1 0.3 21.4 16 + 0 2, dichorionic, diamnioticnegative LCMPC14 0.2 −0.3 0.0 6.8 12 + 5 2, n.a., n.a. negative LCMPC152.2 0.1 14.7 24.8 16 + 0 2, dichorionic, diamniotic T21 positive LCMPC161.1 1.7 0.5 5.4 12 + 5 2, n.a., n.a. negative LCMPC17 0.7 1.4 0.5 16.514 + 2 2, n.a., n.a. negative LCMPC18 0.3 2.6 0.0 18.5 18 + 3 2, n.a.,n.a. negative LCMPC19 −0.2 0.8 0.3 16.6 14 + 0 2, dichorionic,diamniotic negative LCMPC20 −0.7 −0.9 0.1 13.1 15 + 4 2, dichorionic,diamniotic negative LCMPC21 1.0 −0.7 1.2 8.4  9 + 3 2, dichorionic,diamniotic negative LCMPC22 −1.1 −0.2 0.3 5.6 16 + 2 2, monochorionic,n.a. negative LCMPC23 −2.2 2.2 −0.8 20.6 19 + 5 2, monochorionic, n.a.negative LCMPC24 −1.6 −0.4 −0.5 14.7 22 + 2 2, monochorionic, diamnioticnegative LCMPC25 −0.8 −0.2 −1.5 12.1 11 + 5 2, n.a., n.a. negativeLCMPC26 −0.4 −0.6 −1.3 7.5 13 + 0 2, dichorionic, diamniotic negativeLCMPC27 0.5 −0.8 −0.4 16.3 12 + 6 2, n.a., n.a. negative LCMPC28 −1.2−0.3 −0.7 19.4 10 + 1 2, dichorionic, diamniotic negative LCMPC29 −0.80.7 −0.4 14.2 13 + 2 2, monochorionic, n.a. negative LCMPC30 0.7 0.3 0.914.9 12 + 2 2, monochorionic, monoamniotic negative LCMPC31 −0.2 0.3−0.9 19.3 19 + 1 2, dichorionic, diamniotic negative LCMPC32 −1.1 2.5−2.2 11.6 20 + 0 2, dichorionic, diamniotic negative LCMPC33 0.2 2.2−1.6 8.6 11 + 0 2, dichorionic, diamniotic negative LCMPC34 −1.0 1.2 0.015.1 15 + 4 2, dichorionic, diamniotic negative LCMPC35 −0.3 −0.8 −0.319.2 12 + 0 2, dichorionic, diamniotic negative LCMPC36 −1.4 −0.5 −0.813.9 12 + 0 2, dichorionic, diamniotic negative LCMPC37 1.8 −0.7 0.113.8 17 + 6 2, dichorionic, diamniotic negative LCMPC38 −0.1 1.1 −0.713.4 13 + 1 2, dichorionic, diamniotic negative LCMPC39 −1.9 0.2 −2.215.0 17 + 0 2, dichorionic, diamniotic negative LCMPC40 0.6 −0.4 0.816.2 18 + 3 2, dichorionic, diamniotic negative

The reliable detection of foetal aneuploidy in twin pregnancies by NIPTis dependent on a sufficiently high amount of foetal cfDNA from eachfoetus in the maternal blood. Different data and considerations havebeen published on how the lower limit of foetal cfDNA fraction should bedefined to ensure that each twin's contribution is above the detectionthreshold (Leung et al 2013, Prenat Diag 33:675; Qu et al 2007, Am JPathol 170:941; Struble et al 2013, Fetal Diagn Ther December 7 Epubahead of print). This is especially important for dichorionic twinpregnancies with discordant karyotypes. In the study described above,supporting information was used for the definition of the minimum foetalcfDNA fraction for twin pregnancies derived from the Y-chromosomalrepresentation, if only one of the two foetuses is male. Using themethod of the present invention, the total foetal cfDNA fraction can bedetermined, which reflects the summary of foetal cfDNA derived from bothfoetuses. Using the Y-chromosomal representation from the nextgeneration sequencing, the foetal cfDNA amount can be determined formale foetuses (as described in Stumm et al 2014). Thus, in the case ofmixed foetal gender the contributing amount of each foetus can bedetermined by subtraction of the amount of foetal cfDNA determined bythe Y-chromosomal representation from the foetal cfDNA fraction measuredby method of the present invention. The foetal cfDNA fractionsdetermined by the method of the present invention were compared with thevalues obtained from Y-chromosomal reads from next generation sequencingfor cases with known gender (see FIG. 3). There is a correlation of theamount of male specific cfDNA (y axis) to the foetal cfDNA fractionmeasured by method of the present invention (x axis). Thus, for twinpregnancies with male/male gender approximately true is: [y=x], forfemale/male genders it is: [y=0.5x] and for female/female: [y=1]. Thegenders of cases with similar values are male/male and in case ofdiffering values with low Y-chromosomal representation the genders arefemale/female. The intermediate cases, which show about half thepercentage of foetal fraction as Y-chromosomal representation, are ofmixed gender. The data presented in FIG. 3 show that it is not onlypossible to determine the foetal genders using NIPT results for twinpregnancies, but also that the measurement of the amount of foetalfraction of cfDNA determined by the method of the present invention issurprisingly accurate as compared to frequency counting of Y chromosomesequences. On the other hand, these data support the hypothesis thateach foetus of a twin pregnancy contributes roughly about half of thetotal foetal cfDNA fraction. This leads to the conclusion that for twinpregnancies, twice the amount of foetal cfDNA would be required, andthus a recommended minimum foetal fraction of cfDNA for NIPT of a twinpregnancy could be considered to be 8%.

For monochorionic twin pregnancies with concordant genotypes (apart fromrare exceptions, Chen et al 2013, Am J Med Genet A 161A:1817), a foetalcfDNA fraction of 4% would be enough to detect a foetal aneuploidy, justas for single pregnancies. However, for routine laboratory NIPT serviceone major issue speaks against the implication of such different qualitycriteria for mono- and dichorionic pregnancies: the determination ofchorionicity is dependent on the gestational age and the practicalexperience of the physician performing the ultrasound examination. Thechorionicity is clearly detectable in the first trimester of a multiplepregnancy, but in later stages detection becomes more difficult(Sperling et al 2001, Acta Obstet Gynecol Scand 80:287). Therefore, itis a safer strategy to generally define a minimum foetal cfDNA fractionfor twin pregnancies, which is applicable for monochorionic as well asfor dichorionic multiple pregnancies.

Identification of Vanishing Twins:

In two cases of NIPT aneuploidy testing in which the foetal cfDNAfraction was measured using the method of the present invention,identified a trisomy 21 (z-scores 13.5 and 3.4 respectively), but also astriking discrepancy between the total foetal cfDNA fraction measured bythe method of the invention and the cf-Foetal-DNA amount measured byY-chromosome representation were observed.

For case A, two analyses of blood samples (first and back-up samples)estimated the total foetal cfDNA fraction measured the method of thepresent invention was 20.7% and 24.8%, respectively, whereas the foetalcfDNA according to the Y-chromosomal representation from next generationsequencing was 9.2% and 9.3%, respectively. It was speculated, andreported to the physician, that the pregnancy may be a mixed-sex twinpregnancy, who confirmed that a deceased twin had been observed duringultrasound scan at week 10. A further blood sample taken in the thirdtrimester of the pregnancy (38+2) turned out to be negative for trisomy21 and the foetal cfDNA amount measured by Y-chromosomal representationcorrelated with the foetal amount measured by QuantYfeX (21.7% and21.4), which matched the male gender determined by karyotyping of theliving foetus. At birth a foetus papyraceus was found in the placentaltissue from which a sufficient amount of cells could be isolated forcell culture and following GTG banding, a trisomy 21 positive, femalekaryotype was confirmed (47,XX,+21).

For case B, a slightly increased Y-chromosomal representation wasmonitored indicating male specific cf-Foetal-DNA of 3.0% and 2.7%respectively. As the foetal cfDNA fraction estimates measured by themethod of the invention were far above that (13.4% and 10.0%) wehypothesized from this discrepancy in the foetal fraction measured, thattwo foetuses with discordant gender contribute to the foetal fractionand the male foetus being the one affected by trisomy 21. Thissuggestion was derived from the correlation of Y-chromosome specificfoetal cfDNA amount of roughly 3% with the elevated z-score around thecut-off value of 3.0. Since the examination was clearly requested for asingleton pregnancy, the male specific foetal cfDNA was suspected tostem from a vanishing twin—maybe the carrier of a trisomy 21—that waseither not recognized or not indicated on the consent form for NIPT.Thus, the result was reported to be indecisive for chromosome 21 and theconflicting data was reported to the responsible physician, including anotice regarding the potential vanishing twin, for further clarificationvia ultrasound. The responsible physician subsequently confirmed thatthe pregnancy had started as twin and later continued as a singletonpregnancy. The gender of the living and apparently healthy foetus wasconfirmed to be female and thus, the foetal cfDNA that caused theincreased z-score for trisomy 21 can clearly be assigned to a deceasedmale foetus. The pregnancy is still on-going and further analysis ofplacental tissue and blood of the living foetus is not yet possible.

Example 2: Improved Detection Sensitivity Using Two DifferentiallyMethylated Regions Using the Same Detectable Moiety/Moieties for EachDifferentially Methylated Region

The inventors were surprised to observe that a complex and multiplexreaction detecting two DMRs using the same detectable moiety/moietiesfor each of said DMR (as well as two other regions (OR) notdifferentially methylated) was more sensitive to detect foetal cfDNAfraction than previous detection reactions that each detected—inseparate PCR reactions—a single DMR (as well as a single OR) (FIG. 4).

In a method of the present invention, two DMRs (those found in RASSF1Aand TBX3, as described in Example 1) were detected (over 4 dilutions)with the same aliquot of DNA and reaction—effectively simultaneously(using quantitative probe-based (TaqMan) PCR) with two ORs (those foundin RASSF1A and TBX3, as described in Example 1), using: (x) the samedetectable moiety/moieties for each of said DMR; and (y) a detectablemoiety/moieties for said at least one OR that is/are different to thedetectable moiety/moieties used for said DMRs. In comparison, detectionof foetal cfDNA fraction was less sensitive, as shown by detection athigher cycle numbers (Cp), if each DMR (and corresponding OR) wasdetected independently in separate reactions. The regions/markers,primers/probes and detection methodology was substantially as describedin Example 1, except that for the single locus reactions, only the DMRand OR from a given gene (RASSF1A or TBX3) were detected simultaneouslyin a single reaction.

In contrast, detection of foetal cf DNA fraction using a multiplexreaction of the two DMRs using different detectable moieties (eg FAM forthe RASSF1A locus and VIC for the TBX3 locus) is determined to be evenless sensitive, and further is difficult to detect simultaneously withany OR; without being bound by theory, believed due to the highercomplexity of colour compensation, the limited number of separatelydetectable fluorescent markers and/or the “bleaching” effects from somany fluorescent markers being present in the same reaction.

Given the exponential nature of quantitative PCR detection, a highersensitivity of detection (ie lower cycle numbers) would also equate tohigher accuracy of quantification, as the correction to standard curves,and interpolation between data points, would be subject to less errorthan that arising with the amounts of DNA correlating to detection athigher cycle numbers.

Example 3: Detection of an Increased Risk of a Pregnant Woman Sufferingfrom or Developing Preeclampsia (Prophetic Example)

Using a method of the present example, pregnant women are assessed fortheir risk of suffering from or developing preeclampsia as follows.Firstly, a blood sample is collected from the woman for whom such riskto be assessed and total cfDNA extracted from the plasma of such samplesubstantially in accordance with the procedures described in Example 1.Secondly, using a method substantially as described in Example 1, arelative and/or absolute amount of foetal cfDNA and total cfDNA presentin the plasma is determined, where the absolute amount of foetal and/ortotal cfDNA can be expressed as the amount of genome equivalents (“Eq”).Thirdly, such determined amount of cfDNA and/or total cfDNA is comparedto a threshold amount or a reference distribution of amounts, and thewomen is determined to be at increased risk of suffering from ordeveloping preeclampsia if the amount of foetal cfDNA or total cfDNAexceeds such threshold value and/or is an outlier in such distribution.

For example, using published threshold values (Papantoniou et al 2013,Prenat Diag 33:682) if the total cfDNA exceeds an amount of about 7,500Eg/mL plasma or if the foetal cfDNA fraction exceeds an amount of about500 Eg/mL plasma, then the woman is determined to have such an increasedrisk. Such a risk may instead or additional be assessed by considering:(i) the fold-increase (eg 1.5, 3, 3.5 or 4-fold increase) of foetalcfDNA (determined for such woman compared to a threshold amount),factoring into the determination that for later-term pregnancies ahigher fold-increase in foetal cfDNA may be utilised (Zeybek et al 2013,J Obstet Gynaecol Res 39:632); and/or (ii) into which percentile theamount of cfDNA determined from the woman falls, from consideration of areference distribution of amounts determined from low-risk women orwomen who did not suffer from or develop preeclampsia, for example ifthe foetal cfDNA fraction falls within the 90^(th) percentile of such adistribution, then the woman is considered to have an increased risk ofsuffering mild or severe preeclampsia (Jakobsen et al 2013, Transfusion53:1956).

In this example, t detection of a risk is conducted using a computerprogram product that performs the operations represented by FIG. 5.Operation (A) receives signals (1) and (2) representing, respectively,foetal and total cfDNA are used by the computer program product todetermine a parameter (4) that represents the relative and/or absoluteamount of foetal (or total) cfDNA present in the plasma of the woman.This operation may optional receive a signal (3) representing anabsolute amount of standard DNA. A second operation (B) compares suchdetermined parameter (4) against a threshold amount (5) and/or areference population of amounts (6) so as to determine and report (7)whether or not—and based on such comparison—the woman is determined tobe at increase risk of suffering or developing preeclampsia.

Example 4: Detection of Tumour-Associated DNA in Samples from CancerPatients (Prophetic Example)

Methylation of RASSF1A and at least one other DMR such as ER-beta(oestrogen receptor beta), RAR-beta2 (retinoic acid receptor beta 2)and/or Cyclin D2 is used to detect cfDNA derived from a tumour and toassess the risk of women suffering from breast cancer. Specificmethylation at such DMRs is a characteristic of tumour-derived cfDNA,and a method of the present invention is used to detect and to quantifythe amount tumour derived cfDNA in the plasma of women, and thosedetermined to have elevated (or outlying) amounts of tumour-derivedcfDNA are determined to be at increased risk from suffering from ordeveloping breast cancer. Essentially, the process described in Example3 is followed except that DMR2 and OR2 are located in one of ER-beta,RAR-beta2 or Cyclin D2, rather than TBX3. Primers and probes to detectsuch DMR2 and OR2 for use in this embodiment of the present inventionare designable by the person of ordinary skill.

In this example, a similar computer program product as described inExample 3 can be used to asses the risk for a given woman is based onthe amount of tumour-derived cfDNA present in her blood, but in thisexample this parameter is compared against a threshold amount ordistribution of amounts that is derived from a study of the amount oftumour-derived cfDNA present in control and breast-cancer patients; andthose women having an elevated (or outlying) amount of tumour-derivedcfDNA are considered to have an increased risk of suffering from ordeveloping breast cancer.

Example 5: Use of a Method of the Invention in NIPT to Detect Trisomy 21

The inventors surprised to observe that a further adaptation of a methodof the present invention could be used to identify cfDNA samplesobtained from pregnant females that were carrying a trisomy 21 foetus.

In a single multiplex PCR reaction, the amount of a foetal chromosomal21 DNA species present in cfDNA samples obtained from 138 pregnant humanfemales was determined using a method of the invention, and the amountof a foetal chromosomal 12 (as a reference chromosome) DNA speciespresent in the cfDNA sample was also determined using a method of theinvention, and in respect of each pregnant female. The relative amountsof such foetal chromosomal 21 species and such foetal chromosomal 12species was calculated as a ratio, and run-specific z-score analysesusing an internal reference set of euploid samples was conducted. Thosesamples showing a z-score greater than about 3 were determined as“positives”. The second scatter plot of FIG. 8 shows that the outlyingpositive samples are those obtained from the 8 pregnant females of theset of 138 known to carry a foetus with trisomy 21 (“T21”). All eightsamples were successfully classified with no false negative or falsepositive result.

The amount of a foetal chromosomal 21 DNA species was determined byusing, in a method of the present example, a DMR present in the DSCAMgene (Down Syndrome Cell Adhesion Molecule; NCBI Reference Sequence Homosapiens chromosome 21, GRCh38.p2 Primary Assembly: NC_000021.9GI:568815577, region 40010999 to 40847113; SEQ ID No.: 200) located onhuman chromosome 21 and an other region also located between about 300bp and 500 bp of the DMR. The amount of a foetal chromosomal 12 DNAspecies was determined by using, in a method of the present example, aDMR present in the TBX3 gene (as described above) located on humanchromosome 12 and an other region also located between about 10 Kb ofthe DMR. The sequences of the respective DMRs and ORs used are describedin TABLE 7.

TABLE 7 Chromosome 21 and chromosome 12 DMRs and ORs SEQ Chr. ID Chr.location Gene Type Sequence (5′-3′) No. 21 40841691- DSCAM DMRATTGGAAGGTCAgCCA 201 40841781 ATCAGGCGCGGAGCTG CTCCCGG(t)AGCTGCCACCTCCGAGGCGCGC GCCACGCCGGGGTTCC cTcGCGGCTTTGGA 21 40841286- DSCAM ORTCCGTGTGCTCCACCC 202 40841372 TTTGAATTCAGAACGA CATAGTGGATACTCCGTGGGGCTGCTGGAATC TTCCaTTCcCACTGCC TTATCTT 12 114687093- TBX3 DMRAAGGTGCGAACTCCTC 203 114687191 TTTGTCTCTGCGTGcC CGGCGCGCCCCCCTCCCgGTGGGTGATAAAcC CACTCTGGCGCCGGcC ATGCGcTGGgTGATTA ATT 12 114676384-TBX3 OR TGTTcACTGGAGGACT 204 114676454 CATCAGAGGTCCCATT CTCCTTTTTGTGTCTTTCATCAAACACCCTCA tGGACTG Methylation sensitive sites are underlined andlocations of known SNPs are shown by non-capitalisation

cfDNA samples from 138 pregnant human females (including 8 of which wereknown to carry a foetus with T21 were collected, prepared and digestedwith the CpG-methylation sensitive enzymes HhaI, HpaI and BstUI asdescribed in EXAMPLE 1. A multiplex quantitative probe-based PCRreaction of the four separate loci described in TABLE 6 was conducted onreplicates (n=6) of each such sample as described in EXAMPLE 1, exceptthat the PCR buffer used was PerfeCTa MultiPlex qPCR ToughMix (QuantaBioSciences) and using the PCR primers and labelled probes (withquenchers) as set forth in TABLE 8.

TABLE 8 Primer and probes SEQ ID Chr. Region ComponentSequence (5′-3′)** No.* 21 DSCAM Chr21DMR- ATTGGAAGGTCAGCCAAT 205 DMRFor CAGG Chr21DMR- TCCAAAGCCGCGAGGGAA 206 Rev C Chr21DMR-[LCCyan500]-CGCCTC 207 Probe GGAGGTGGCAGCTC- [BHQ1] 21 DSCAM Chr21OR-TCCGTGTGCTCCACCCTT 208 Other For TG region Chr21OR- AAGATAAGGCAGTGGGAA209 Rev TGGAAG Chr21OR- [Cy5]-CCAGCAGCCCCA 210 Probe CGGAGTATCC-[BHQ3]12 TBX3 Chr12DMR- AAGGTGCGAACTCCTCTT 211 DMR For TGTC Chr12DMR-AATTAATCACCCAGCGCA 212 Rev TGGC Chr12DMR- [6FAM]-CCCCTCCCGGT 213 ProbeGGGTGATAAACC- [Eclipse] 12 TBX3 Chr12OR- TGTTCACTGGAGGACTCA 214 OtherFor TC region Chr12OR- CAGTCCATGAGGGTGTTT 215 Rev G Chr12OR-[LCRed610]-AGGTCCC 216 Probe ATTCTCCTTTTTGTGTCT TTC-[BBQ650] *Onlynucleotide sequence listed, without dyes/quenchers **The dyes/quenchersused for each probe are shown in “[]” parentheses

The format of such assay is generally as depicted in FIG. 6, where theDMR1 and OR1 of such figure are located in the DSCAM gene of humanchromosome 21, and the DMR1′ and OR1′ of such figure are located in theTBX3 gene of human chromosome 12 (in this example, each chromosome wasdetected with only one DMR/OR pair, and hence the optional second pairsrepresented by (2) and (2′) in such figure are not included in thepresent example). As will be observed from TABLE 8, the probe for thechromosome 21 DMR is labelled differently from the probe for thechromosome 12 DMR (and each of the ORs are differently labelled). Thisenables the foetal fraction of cfDNA for each chromosome-specific DNAspecies to be separately calculated by relative quantification of thechromosome-specific DMR to the respective chromosome-specific OR, andthe absolute total cfDNA amount for each chromosome was calculated byabsolute quantification of signals for the respective OR from the samplecompared to signals for the respective OR obtained from the dilutionseries of standard DNA of known concentration (as described inEXAMPLE 1) provided in each qPCR plate (run) as the test samples. Suchrelative and absolute quantifications were conducted using LightCycler480 Software release 1.5.0 (Roche). The mean [n=6 replicates]sample-specific ratio of the % foetal cfDNA chromosome 21 DNA speciesand the % foetal cfDNA chromosome 12 DNA species was calculated for eachsample, and for each plate the overall mean and standard deviation ofsuch ratios for the known euploid samples were calculated; from whichparameters all test samples on such qPCR plate (run) were individuallyanalysed to give a Z-score (on such on a plate-by-plate basis) for eachtest sample (including those test samples known to be trisomy 21). Thesecond scatter plot in FIG. 8 displays the z-score (calculated on aplate-by-plate basis) for each test sample showing separation of alltrisomy samples from the euploid samples.

Example 6: Iterative z-Score Analysis to Detect Trisomy 21 in NIPTwithout Reference to Known Internal Euploid Standards

By the application of an iterative z-score approach, the inventors wereable to identify all known Trisomy 21 samples from the test sampleswithout reference to the known euploid samples in the estimation of meanand standard deviations in the z-score analysis.

The data from each run (qPCR plate) of samples analysed in EXAMPLE 5 wasre-analysed as follows. Firstly, the mean ration of chromosome 21 toreference chromosome of each replicate of the samples (n=6) wascalculated, and for all samples present in such run (plate) an overallmean and standard deviation was calculated without reference to whethera sample was known to be euploid or trisomy. The first mean ratio foreach sample is shown in the first scatter plot of FIG. 8 (with theeuploid and T21 samples plotted with different symbols) for all samplesacross all runs. Secondly, based on such run-specific means and standarddeviation, a (run specific) z-score for each sample present in the runwas calculated. The third scatter plot of FIG. 8 shows such maskedz-scores for all samples across all runs. Thirdly, those samples showinga z-score of greater than about 1.9 were removed from the data set, asecond mean and standard deviation was calculated on the data in respectof the remaining samples in the data set and used to conduct a secondz-score analysis of all samples in the set. The fourth scatter plot ofFIG. 8 shows the z-scores for all samples across all runs following suchfirst iterative elimination. Fourthly, those samples showing a z-scorefrom the first iterative elimination of greater than about 1.9 were alsoremoved from the data set, a third mean and standard deviation wascalculated on the data in respect of the remaining samples in the dataset and used to conduct a third z-score analysis of all samples in theset. The fifth scatter plot of FIG. 8 shows the z-scores for all samplesacross all runs following such second iterative elimination and displayscomplete separation of the euploid and T21 samples (represented byz-scores greater than about 3.0); without reference to any of thesamples a-priori known to be euploid.

In view of the above, it will be appreciated that the present inventionalso relates to the following items:

-   1. A method for detecting in a sample from an individual an amount    of a species of DNA originating from cells of a given type, which    sample comprises said species of DNA in admixture with differently    methylated DNA not originating from cells of said type; said method    comprising the steps:    -   (a) treating the DNA present in said sample with a reagent that        differentially modifies methylated and non-methylated DNA;    -   (b) detecting in said sample the presence of methylation in said        species of DNA at two or more differentially methylated regions        (DMRs) that are differently methylated between said species of        DNA and the DNA not originating from cells of said type, the        modification of DNA of such DMRs by said reagent is sensitive to        methylation of DNA, wherein the presence of methylated DNA at        one or more of said DMRs indicates the presence of said amount        of species of DNA in said sample and the absence of methylated        DNA at said DMRs indicates the absence of said species of DNA in        said sample; and    -   (c) detecting an amount of total DNA present in said sample        using at least one other region that is not differently        methylated between said species of DNA and the DNA not        originating from cells of said type, the modification of which        region(s) by said reagent is insensitive to methylation of DNA,    -   wherein, said detection in step (b) and said detection in        step (c) are made using the same aliquot of DNA of said sample,        and in the same vessel, and effectively simultaneously for such        DMRs and other region(s), and using: (x) the same detectable        labels(s) for each of said DMRs; and (y) a different detectable        label(s) for said other region(s).-   2. The method of item 1, wherein prior to or as part of said    detection in step (b) and/or step (c), each DNA region comprising    said DMRs and/or said other region(s), respectively, is(are)    amplified.-   3. The method of item 1 or 2, wherein each detectable label used in    step (b) and/or step (c) is independently selected from the group    consisting of: fluorescent, protein, small molecule or radioactive    label.-   4. The method of any one of items 1 to 3, wherein said detection in    step (b) comprises multiplex real-time probe-based quantitative    probe-based PCR using at least two labelled probes each of which    specific for one of said DMRs.-   5. The method of any one of items 1 to 4, wherein said detection in    step (c) comprises real-time quantitative PCR using at least one    labelled probe specific for one of said other region(s).-   6. The method of any one of items 1 to 5, wherein said other region    is located between about 20 bp and about 20 kb upstream or    downstream of, and/or within the same gene as, at least one of said    DMRs.-   7. The method of any one of items 1 to 6, wherein said detection in    step (c) comprises using at least two of said other regions;    preferably wherein, the number of said other regions is the same as    the number of DMRs used in step (b); more preferably wherein, one of    said other regions is located between about 20 bp and about 20 kb    upstream or downstream of a DMR used in step (b) and each other of    the said other regions is located between about 20 bp and about 20    kb upstream or downstream of another of said DMRs.-   8. The method of item 7, wherein said detection in step (c) is made    using: (x) the same detectable label(s) for each of said other    regions or (y) a different detectable label(s) for each of said    other regions.-   9. The method of item 7 or 8, wherein said detection in step (c)    comprises multiplex real-time quantitative probe-based PCR using at    least two labelled probes each of which is specific for one of said    other regions.-   10. The method of any one of items 1 to 9, wherein said detection in    step (c) and said detection in step (b) are made using the same    aliquot of DNA of said sample, and in the same reaction/detection    vessel, and effectively simultaneously with each other, and by    multiplex real-time quantitative probe-based PCR using at least one    labelled probe specific for each of the said DMRs and other    region(s).-   11. The method any one of items 1 to 10, wherein said species of DNA    originates from cells of a foetus and/or the placenta of a foetus    and said sample is from a pregnant female; preferably wherein, said    species of DNA is circulating cell-free DNA and said sample is a    blood fraction such as plasma or serum.-   12. The method of item 11, wherein said DMRs comprises at least one,    preferably at least two, methylation site(s) specific for said    reagent, and at least one of said DMRs is located in a portion of    the genome and/or gene selected from the group consisting of:    RASSF1A, TBX3, HLCS, ZFY, CDC42EP1, MGC15523, SOX14 and SPN;    preferably wherein,    -   each of said DMRs is located in a portion of the genome and/or        gene selected from the group consisting of: RASSF1A, TBX3, HLCS,        ZFY, CDC42EP1, MGC15523, SOX14 and SPN; and/or    -   at least one of said DMRs is located between about positions        4,700 bp and 5,600 bp of RASSF1A or about positions 1,660 bp and        2,400 bp of TBX3; more preferably wherein,    -   said two or more DMRs comprise those located between about        positions 4,700 bp and 5,600 bp of RASSF1A and about positions        1,660 bp and 2,400 bp of TBX3.-   13. The method of item 11 or 12, wherein said other region is    located in a portion of the genome and/or gene selected from the    group consisting of: GAPDH, beta-actin, ALB, APOE, RNASEP, RASSF1A,    TBX3, HLCS, ZFY, CDC42EP1, MGC15523, SOX14 and SPN; preferably    wherein,    -   said other region comprises a region without a methylation site        specific for said reagent and said locus is located in the genes        RASSF1A or TBX3, more preferably wherein,    -   two or more of said other regions are used in detection step (c)        and comprise those located between about positions 14,220 bp and        13,350 bp of RASSF1A and about positions 12,400 bp and 13,000 bp        of TBX3.-   14. The method any one of items 11 to 13, wherein said pregnant    female is susceptible to a pregnancy-associated medical condition;    preferably wherein, said pregnancy-associated medical condition is    selected from the group consisting of: preeclampsia, preterm labour,    intrauterine growth retardation and vanishing twin.-   15. The method of any one of items 1 to 10, wherein said species of    DNA originates from a cell type associated with a medical condition;    preferably wherein, said medical condition is one selected from the    group consisting of: a cell proliferative disorder, an    infection/infectious disease, a wasting disorder, a degenerative    disorder, an (auto)immune disorder, kidney disease, liver disease,    inflammatory disease acute toxicity, chronic toxicity, myocardial    infarction, and a combination of any of the forgoing; more    preferably wherein, said species of DNA is circulating cell-free DNA    and said sample is a blood fraction such as plasma or serum.-   16. The method of item 15, wherein said species of DNA originates    from cells of a tumour; preferably wherein, said tumour is a    carcinoma or cancer of an organ selected from the group consisting    of: liver, lung, breast, colon, oesophagus, prostate, ovary, cervix,    uterus, testis, brain, bone marrow and blood.-   17. The method of item 16, wherein said DMRs comprises at least one,    preferably at least two, methylation site(s) specific for said    reagent, and at least one of said DMR is located in a portion of the    genome and/or a gene selected from the group consisting of: a tumour    suppressor gene, p16, SEPT9, RASSF1A, GSTP1. DAPK, ESR1, APC,    HSD17B4 and H1C1; preferably wherein, one of said two or more DMRs    is located in RASSF1A; more preferably wherein, one of said two or    more DMRs is located between about positions 4,700 bp and 5,600 bp    of RASSF1A; and/or more preferably wherein, said other region is    located between about positions 14,220 bp and 13,350 bp of RASSF1A.-   18. The method of any one of items 1 to 17, wherein said sample is a    tissue sample or a sample of biological fluid; preferably wherein,    said sample is a sample of biological fluid selected from the group    consisting of: whole blood, a blood fraction, urine, saliva, sweat,    ejaculate, tears, phlegm, vaginal secretion, vaginal wash and    colonic wash; more preferably wherein, said sample is a plasma or    serum sample.-   19. The method of any one of items 1 to 18, wherein said reagent    that differentially modifies methylated and non-methylated DNA    comprises bisulphite.-   20. The method of any one of items 1 to 18, wherein said reagent    that differentially modifies methylated and non-methylated DNA    comprises an agent that selectively digests unmethylated over    methylated DNA, preferably wherein, said agent comprises:    -   at least one methylation sensitive enzyme;    -   at least one methylation sensitive restriction enzyme; and/or    -   an agent selected from the group consisting of: AatII, AciI,        AcII, AfeI, AgeI, AgeI-HF, AscI, AsiSI, AvaI, BceAI, BmgBI,        BsaAI, BsaHI, BsiEI. BsiWI, BsmBI, BspDI, BsrFI, BssHII, BstBI,        BstUI, ClaI, EagI, FauI, FseI, FspI, HaeII, HgaI, HhaI, HinP1I,        HpaII, Hpy99I, HpyCH4IV, KasI, MluI, NaeI, NarI, NgoMIV, NotI,        NotI-HF, NruI, Nt.BsmAI, Nt.CviPII, PaeR7I, PluTI, PmlI, PvuI,        PvuI-HF, RsrII, SacII, SalI, SalI-HF, SfoI, SgrAI, SmaI, SnaBI,        TspMI and ZraI.-   21. The method of any one of items 1 to 20, wherein each of said    detection steps comprises quantitative detection and said detected    amount of said species of DNA is expressed as a relative    concentration of said species of DNA to the total DNA in said    sample.-   22. The method of any one of items 1 to 20, further comprising the    steps:    -   detecting an amount of total DNA in a standard sample of DNA of        known amount using the same other regions(s) as used in step        (c); and    -   comparing the signal detected from said standard sample of DNA        to the signal detected in step (c).-   23. The method of item 22, wherein each of said detection steps    comprises quantitative detection and said detected amount of said    species of DNA is expressed as an absolute amount of said species of    DNA in said sample.-   24. The method of item 21 or 23, further comprising the step:    -   comparing the amount of said species of DNA detected with a        threshold amount and/or reference distribution of amounts,        wherein: (x) an increase in, or outlying of, the amount of said        species of DNA indicates an increased risk of the individual        suffering from or developing a medical condition; and/or (y) an        amount of said species of DNA in excess to said threshold, or        outlying from said distribution, indicates that a diagnosis for        an abnormality in the said species of DNA present in said sample        may be performed on, preferably a separate aliquot of DNA of,        said sample.-   25. The method of any one of items 21 to 24, further comprising the    step:    -   performing on, preferably with a separate aliquot of DNA of,        said sample, a diagnosis for an abnormality in said species of        DNA present in said sample; preferably wherein, said species of        DNA originates from cells of a foetus and/or the placenta of a        foetus, said sample is from a pregnant female and said diagnosis        is a prenatal diagnosis.-   26. The method of item 25, wherein said diagnosis comprises a step    that uses a detection technology selected from the group consisting    of: DNA sequencing, SNP analysis, digital PCR and hybridisation;    preferably wherein, said detection technology is massively parallel    sequencing of DNA; more preferably wherein said detection technology    is massively parallel sequencing of random and/or enriched DNA.-   27. The method of item 25 or 26, wherein:    -   (x) said species of DNA originates from cells of a foetus and/or        the placenta of a foetus, said sample is from a pregnant female        and said abnormality is a genetic mutation or a chromosomal        abnormality, such as a chromosomal trisomy, associated with a        foetal abnormality and/or a congenital disorder; preferably        wherein:        -   said genetic mutation is selected from the group consisting            of: colour blindness, cystic fibrosis, hemochromatosis,            haemophilia, phenylketonuria, polycystic kidney disease,            sickle-cell and disease, Tay-Sachs disease; and/or        -   said chromosomal abnormality is selected from the group            consisting of: a trisomy (such as trisomy 21, trisomy 18, or            trisomy 13), a sex-chromosome abnormality (such as Turners            syndrome, Klinefelter syndrome, [Noonan syndrome,] Triple X            syndrome, XXY syndrome, or Fragile X syndrome or XYY            syndrome or XXYY syndrome), a chromosomal deletion (such as            Prader-Willi syndrome, Cris-du-chat syndrome,            Wolf-Hirschhorn syndrome, or 22q11 deletion syndrome,            Duchene muscular dystrophy), Beckwith-Wiedemann syndrome,            Canvan syndrome, and neurofibromatosis; or    -   (y) said species of DNA originates from cells of a tumour and        said abnormality is a genetic mutation or a chromosomal        abnormality associated with the diagnosis, prognosis or        predictive treatment of a carcinoma or cancer; preferably        wherein:        -   said genetic mutation is selected from the group consisting            of: a mutation in a tumour suppressor gene (such as TP53            (p53), BRCA1, BRCA2, APC or RB1), a mutation in a            proto-oncogene (such as RAS, WNT, MYC, ERK, or TRK) and a            DNA repair gene (such as HMGA1, HMGA2, MGMT or PMS2); and/or        -   said chromosomal abnormality is a translocation (such as            t(9;22)(q34;q11) [ie, Philadelphia chromosome or BCL-ABL],            t(8;14)(q24;q32), t(11;14)(q13;q32), t(14;18)(q32;q21),            t(10;(various))(q11;(various)), t(2;3)(q13;p25),            t(8;21)(q22;q22), t(15;17)(q22;q21), t(12;15)(p13;q25),            t(9;12)(p24;p13), t(12;21)(p12;q22), t(11;18)(q21;q21),            t(2;5)(p23;q35), t(11;22)(q24;q11.2-12), t(17;22),            t(1;12)(q21;p13), t(X;18)(p11.2;q11.2), t(1;19)(q10;p10),            t(7,16)(q32-34;p11), t(11,16)(p11;p11), t(8,22)(q24;q11) or            t(2;8)(p11;q24)).-   28. The method of item 11, wherein said DMR(s) is/are    hypermethylated in foetal DNA and hypo methylated in maternal DNA.-   29. The method item 28, wherein said DMR(s) comprises at least one,    preferably at least two, methylation site(s) specific for said    reagent, and at least one of said DMR(s) is located in a region    and/or gene selected from the list(s) consisting of one disclosed in    WO 2011/034631 as being hypermethlyated in foetal DNA relative to    maternal DNA, including SEQ ID NOs 1-59, 90-163, 176, 179, 180, 184,    188, 189, 190, 191, 193, 195, 198, 199, 200, 201, 202, 203, 205,    206, 207, 208, 209, 210, 211, 212, 213, 214, 221, 223, 225, 226,    231, 232, 233, 235, 239, 241, 257, 258, 259, and/or 261 of WO    2011/034631.-   30. The method of item 29, wherein at least one of said DMR(s) is    located in a region and/or gene selected from the list consisting    of: SEQ ID NOs 1-39, 176, 179, 180, 184, 188, 189, 190, 191, 193,    195, 198, 199, 200, 201, 202, 203, 205, 206, 207, 208, 209, 210,    211, 212, 213, 214, 221, 223, 225, 226, 231, 232, 233, 235, 239,    241, 257, 258, 259, and/or 261 of WO 2011/034631, preferably    selected from the list consisting of: SEQ ID No NOs 33-39, 176, 179,    180, 184, 188, 189, 190, 191, 193, 195, 198, 199, 200, 201, 202,    203, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 221, 223,    225, 226, 231, 232, 233, 235, 239, 241, 257, 258, 259, and/or 261 of    WO 2011/034631.-   31. The method item 11 or 28, wherein said DMR(s) comprises at least    one, preferably at least two, methylation site(s) specific for said    reagent, and at least one of said DMR(s) is located in a region    and/or gene disclosed in WO 2011/092592, including on selected from    the list(s) consisting of: EP1, EP2, EP3, EP4, EP5, EP6, EP7, EP8,    EP9, EP10, EP11 and EP12 [SEQ ID NOs 33-44] of WO 2011/092592.-   32. The method item 11 or 28, wherein said DMR(s) comprises at least    one, preferably at least two, methylation site(s) specific for said    reagent, and at least one of said DMR(s) is located in a region    and/or gene selected from the list consisting of: AIRE, SIM2, ERG    and VAPA-APCDDI, or is HLCS.-   33. The method of any one of items 11 or 28 to 32, wherein at least    one of said DMR(s):    -   is located on a human chromosome selected from the list        consisting of: chromosome 21, chromosome 18, chromosome 13,        X-chromosome and Y-chromosome, preferably chromosome 21,        chromosome 18, chromosome 13, most preferably chromosome 21;        and/or    -   comprises at least one, preferably at least two, methylation        site(s) specific for said reagent, and said DMR is located in a        regions and/or gene selected from the list consisting of: maspin        [preferably a portion of the maspin (aka “SERPINB5”) gene that        described in EP 1 751 307 as being differentially methylated        between a foetus and its mother], CGI137, PDE9A, PPP1R2P2,        Similarity to Fem1A (C. elegans), CGI009, CBR1, DSCAM, C21orf29        and CGI13.-   34. The method of item 11 or 28, wherein at least one of said DMR(s)    is located in a region and/or gene selected from the list consisting    of: RASSF1A, TBX3, ZFY, CDC42EP1, MGC15523, SOX14 and SPN.-   35. The method of item 11 or 28, wherein at least one of said DMR(s)    is located in a region and/or gene selected from the list consisting    of: SEQ ID NOs: 40-59 and 90-163 of WO 2011/034631.-   36. The method of any one of items 11, 28, 29, 34 or 35, wherein at    least one of said DMR(s):    -   is located on a human chromosome selected from the list        consisting of: chromosome 1 to 12, chromosome 14 to 17,        chromosome 19, chromosome 20 chromosome 22 and chromosome 23;        and/or    -   comprises at least one, preferably at least two, methylation        site(s) specific for said reagent, and said DMR is located in a        regions and/or gene selected from the list consisting of: CD48,        FAIM3, ARHGAP25, SELPLG, APC, CASP8, RARB, SCGB3A1, DAB2IP,        PTPN6, THY1, TMEFF2 and PYCARD.-   37. The method of any of items 1 to 36, wherein a plurality of    species of DNA are detected in said sample; preferably wherein, two    species of DNA are detected in said sample.-   38. The method of item 37, wherein:    -   in at least one detection step (b):        -   the presence of methylated DNA at a first set of two or more            DMRs is used to indicate the presence of an amount of a            first species of DNA in said sample and the absence of            methylated DNA at said first set of DMRs indicates the            absence of said first species of DNA in said sample;            preferably wherein, said first species of DNA originates            from cells of a foetus and/or the placenta of a foetus, said            sample is from a pregnant female and at least one of said            first set of DMRs is one set forth in any one of items 30 to            33; and        -   the presence of methylated DNA at a second set of two or            more DMRs is used to indicate the presence of an amount of a            second species of DNA in said sample and the absence of            methylated DNA at said second set of DMRs indicates the            absence of said second species of DNA in said sample;            preferably wherein, said second species of DNA originates            from cells of a foetus and/or the placenta of a foetus, said            sample is from a pregnant female and at least one of said            second set of DMRs is one set forth any one of items 34 to            36; and    -   in at least one detection step (c):        -   a first amount of total DNA present in said sample is            detected using a first region that is not differently            methylated between said first species of DNA and the DNA not            originating from cells of said type, the modification of            which first other region by said reagent is insensitive to            methylation of DNA, wherein said first other region is            located between about 20 bp and about 20 kb upstream or            downstream of at least one of said first set of DMRs; and        -   a second amount of total DNA present in said sample is            detected using a second region that is not differently            methylated between said second species of DNA and the DNA            not originating from cells of said type, the modification of            which second other region by said reagent is insensitive to            methylation of DNA, wherein said second other region is            located between about 20 bp and about 20 kb upstream or            downstream of at least one of said second set of DMRs.-   39. The method of item 38, wherein said detection in step (b) of    said first and second set of DMRs and said detection in step (c) of    said first and second other regions are made using the same aliquot    of DNA of said sample, and in the same reaction/detection vessel,    and effectively simultaneously for such DMRs and other regions, and    using: (x) a different detectable label(s) for each of said first    and second set of DMRs DMRs; and (y) further different detectable    label(s) for each of said first and second other regions.-   40. The method of item 39, wherein said detection in step (c) and    said detection in step (b) are made by multiplex real-time    quantitative probe-based PCR using at least one labelled probe    specific for each of said DMRs and other regions.-   41. The method of any one of items 37 to 40, wherein said agent    comprises at least one methylation sensitive restriction enzyme.-   42. The method of any one of items 38 to 41, wherein each of said    detection steps comprises quantitative detection and said detected    amount of each of said species of DNA is expressed as a relative    concentration of said species of DNA to the respective amount total    DNA detected in said sample from the respective other region.-   43. The method of any one of items 38 to 41, further comprising the    steps:    -   detecting an amount of total DNA in a standard sample of DNA of        known amount using the same other regions as used in step (c);        and    -   comparing the signal detected from said standard sample of DNA        to the respective signal detected in step (c) for each of the        other regions.-   44. The method of item 43, wherein each of said detection steps    comprises quantitative detection and said detected amount of said    species of DNA is expressed as an absolute amount of each or said    species of DNA in said sample.-   45. The method of item 42 or 44, further comprising the step:    -   determining the relative amount, preferably a ratio, of: (x)        said first species of DNA detected with the first set of two or        more DMRs; and (y) said second species of DNA detected with the        second set of two or more DMRs; and    -   comparing said relative amount or ratio with a threshold and/or        reference distribution of amount(s) or ratio(s), wherein: a        relative amount or ratio that is higher or lower than said        threshold and/or reference distribution of amount(s) or ratio(s)        indicates the presence of an abnormality in said first and/or        second species of DNA present in said sample; preferably        wherein, said abnormality is a chromosomal abnormality; more        preferably wherein, said chromosomal abnormality is associated        with a foetal abnormality and/or congenital disorder; yet more        preferably wherein, said chromosomal abnormality is selected        from the group consisting of: a trisomy (such as trisomy 21,        trisomy 18, or trisomy 13), a sex-chromosome abnormality (such        as Turners syndrome, Klinefelter syndrome, [Noonan syndrome,]        Triple X syndrome, XXY syndrome, or Fragile X syndrome or XYY        syndrome or XXYY syndrome), a chromosomal deletion (such as        Prader-Willi syndrome, Cris-du-chat syndrome, Wolf-Hirschhorn        syndrome, or 22q11 deletion syndrome, Duchene muscular        dystrophy), Beckwith-Wiedemann syndrome, Canvan syndrome, and        neurofibromatosis; most preferable wherein, said chromosomal        abnormality is a trisomy, such as one selected from the list        consisting of trisomy 21, trisomy 18, or trisomy 13.-   46. A method for detecting a chromosomal aneuploidy in a foetus    carried by a pregnant female, said method comprising the steps:    -   (A) Determining, using a method as set forth in any one of the        items above, in a sample taken from said pregnant female the        amount of a first species of DNA that originates from cells of a        foetus and/or the placenta of a foetus, wherein said first        species of DNA is located on a chromosome relevant to the        chromosomal aneuploidy or within a section of a chromosome        relevant to the chromosomal aneuploidy, and wherein said first        species of DNA that originates from cells of a foetus and/or the        placenta of a foetus is distinguished from its counterpart of        maternal origin in the sample due to differential DNA        methylation;    -   (B) Determining, using a method as set forth in any one of the        items above, the amount of a second species of DNA that        originates from cells of a foetus and/or the placenta of a        foetus in said sample, wherein said second species of DNA is        located on a reference chromosome, and wherein said second        species of DNA that originates from cells of a foetus and/or the        placenta of a foetus is distinguished from its counterpart of        maternal origin in the sample due to differential DNA        methylation;    -   (C) determining the relative amount, preferable the ratio, of        the amounts from (A) and (B); and    -   (D) comparing said relative amount or ratio with a threshold        and/or reference distribution of amount(s) or ratio(s), wherein:        a relative amount or ratio that is higher or lower than said        threshold and/or reference distribution of amount(s) or ratio(s)        indicates the presence of the chromosomal aneuploidy in the        foetus.-   47. A method for detecting an increased risk of an individual    suffering from or developing a medical condition; said method    comprising the steps:    -   (i) conducting the method of item 21 or 23; and    -   (ii) comparing the amount of said species of DNA detected with a        threshold amount and/or a reference distribution of amounts,    -   wherein an increase in, or outlying of, the amount of said        species of DNA indicates an increased risk of the individual        suffering from or developing said medical condition.-   48. A composition comprising:    -   two pairs of PCR primers, each pair for amplifying one of said        two of more DMRs as set forth in any of items 1 to 47;    -   one pair of PCR primers for amplifying said other region as set        forth in any of items 1 to 47;    -   two labelled probes as set forth in item 4; and    -   one labelled probe as set forth in item 5.-   49. The composition of item 48, further comprising:    -   a further pair of PCR primers for amplifying a second other        region as set forth in any of items 9 to 47; and    -   a further labelled probe as set forth in item 9.-   50. A kit comprising:    -   the primers and probes as set forth in item 48 or 49; and    -   optionally, further comprising: (i) a printed manual or computer        readable memory comprising instructions to use said primers and        probes to practice a method of any one of items 1 to 47 and/or        to produce or use the composition of item 48 or 49; and/or (ii)        one or more other item, component or reagent useful for the        practice of a method of any one of items 1 to 47 and/or the        production or use of the composition of item 48 or 49, including        any such item, component or reagent disclosed herein, such as        the reagent that differently modifies methylated and        non-methylated DNA as set forth in any one of items 1 to 47.-   51. A computer program product comprising a computer readable medium    encoded with a plurality of instructions for controlling a computing    system to perform and/or manage an operation for determining: (x) an    increased risk of an individual suffering from or developing a    medical condition and/or (y) if a diagnosis for an anomaly in a    species of DNA originating from cells of a given type may be    performed, in each case from a sample from an individual comprising    a species of DNA originating from cells of a given type in admixture    with differently methylated DNA not originating from cells of said    type, the DNA in present in said sample being treated with a reagent    that differentially modifies methylated and non-methylated DNA as    set forth in any one of items 1 to 47; said operation comprising the    steps of:    -   receiving: (i) one signal representing the essentially        simultaneous quantitative detection of methylation at two or        more DMRs as set forth in step (b) of any one of items 1 to 47;        and (ii) one signal representing the essentially simultaneous        quantitative detection of total DNA using at least one other        region as set forth in step (c) any of items 1 to 47;    -   determining a parameter from the signals (i) and (ii), wherein        the parameter represents a quantitative amount of said species        of DNA;    -   comparing the parameter to with a threshold amount and/or        reference distribution of amounts; and    -   based on such comparison, determining a classification of        whether, respectively, (x) an increased risk of an individual        suffering from or developing a medical condition exists;        and/or (y) a diagnosis for an anomaly in a species of DNA        originating from cells of a given type may be performed.-   52. The computer program product of item 51, wherein said operation    further comprises the steps:    -   receiving a further signal representing the quantitative        detection of total DNA in a standard sample of DNA as set forth        in item 22; and    -   comparing said signal with the signal set forth in (ii) of item        51, so as to determine said parameter that represents an        absolute quantitative amount of said species of DNA.-   53. The computer program product of item 51 or 52, wherein said    operation is for determining if a diagnosis for an anomaly in said    species of DNA may be performed, and further comprises the step of    determining from said parameter a number of random and/or enriched    DNA molecules to be sequenced from, preferably from a separate    aliquot of DNA of, said sample as part of said diagnosis.-   54. The computer program product of item 51 or 52, wherein said    operation further comprises the steps:    -   receiving: (i) one signal representing the quantitative        detection of methylation at a second set of two or more DMRs as        set forth in step (b) of any one of items 38 to 46; and (ii) one        signal representing the quantitative detection of total DNA        using a second other region as set forth in step (c) any of        items 38 to 46;    -   determining a second parameter from the signals (i) and (ii),        wherein the parameter represents a quantitative amount of said        second species of DNA;    -   determining the relative amount, preferable the ratio, of said        parameter and said second parameter;    -   comparing said relative amount or ratio with a threshold and/or        reference distribution of amount(s) or ratio(s); and    -   based on such comparison, determining a classification of        whether an abnormality in said species of DNA or second species        of DNA present in said sample; preferably wherein, said        abnormality is a chromosomal abnormality; more preferably        wherein, said chromosomal abnormality is associated with a        foetal abnormality and/or congenital disorder; yet more        preferably wherein, said chromosomal abnormality is selected        from the group consisting of: a trisomy (such as trisomy 21,        trisomy 18, or trisomy 13), a sex-chromosome abnormality (such        as Turners syndrome, Klinefelter syndrome, [Noonan syndrome,]        Triple X syndrome, XXY syndrome, or Fragile X syndrome or XYY        syndrome or XXYY syndrome), a chromosomal deletion (such as        Prader-Willi syndrome, Cris-du-chat syndrome, Wolf-Hirschhorn        syndrome, or 22q11 deletion syndrome, Duchene muscular        dystrophy), Beckwith-Wiedemann syndrome, Canvan syndrome, and        neurofibromatosis; most preferable wherein, said chromosomal        abnormality is a trisomy, such as one selected from the list        consisting of trisomy 21, trisomy 18, or trisomy 13.-   55. A method for detecting in a sample from an individual an amount    of a species of DNA originating from cells of a given type, which    sample comprises said species of DNA in admixture with    differentially methylated DNA not originating from cells of said    type; said method comprising the steps:    -   (a) treating the DNA present in said sample with a reagent that        differentially modifies methylated and non-methylated DNA; and    -   (b) detecting in said sample the presence of methylation in said        species of DNA at two or more DMRs that are differently        methylated between said species of DNA and the DNA not        originating from cells of said type the modification of DNA of        such DMRs by said reagent is sensitive to methylation of DNA,        wherein the presence of methylated DNA at one or more of said        DMRs indicates the presence of said amount of species of DNA in        said sample and the absence of methylated DNA at said DMRs        indicates the absence of said species of DNA in said sample,    -   wherein, said detection in step (b) is made using the same        aliquot of DNA of said sample, and in the same        reaction/detection vessel, and effectively simultaneously for        such DMRs, and using (x) multiplex real-time quantitative PCR;        and (y) at least two labelled probes each of which specific for        one of said DMRs and that are labelled with the same detectable        label(s) for each of said DMRs; preferably wherein, said reagent        comprises agent as set forth in item 20.

The invention claimed is:
 1. A method for quantitatively detecting in asample from an individual an amount of a species of DNA originating fromcells of a given type, which sample comprises said species of DNA inadmixture with differently methylated DNA not originating from cells ofsaid type, wherein said species of DNA originates from cells of a fetusand/or the placenta of a fetus and said sample is from a pregnantfemale; said method comprising the steps: (a) treating the DNA presentin said sample with a reagent that differentially modifies methylatedand non-methylated DNA; (b) quantitatively detecting in said sample thepresence of methylation in said species of DNA at two or moredifferentially methylated regions (DMRs) that are differently methylatedbetween said species of DNA and the DNA not originating from cells ofsaid type, the modification of DNA of such DMRs by said reagent issensitive to methylation of DNA, wherein the presence of methylated DNAat one or more of said DMRs indicates the presence of said amount ofspecies of DNA in said sample and the absence of methylated DNA at saidDMRs indicates the absence of said species of DNA in said sample,wherein said DMRs are hyper-methylated in fetal DNA and hypo-methylatedin maternal DNA, and wherein said DMRs comprise at least one, or atleast two, methylation site(s) specific for said reagent, and at leastone of said DMRs is located in a region and/or gene selected from thegroup consisting of SEQ ID NOs 15-187 or located in a region and/or geneselected from the group consisting of: SEQ ID NOs 188-199; and (c)quantitatively detecting an amount of total DNA present in said sampleusing at least one other region, wherein there is no detectabledifference between modification by said reagent at the other region(s)of DNA originating from cells of a fetus and/or the placenta of a fetusas compared to the other region(s) of maternal DNA, wherein, saiddetection in step (b) and said detection in step (c) are made using thesame aliquot of DNA of said sample, and in the same reaction/detectionvessel, and effectively simultaneously for such DMRs and otherregion(s), and using the same detectable label(s) for each of said DMRsand a different detectable label(s) for said other region(s).
 2. Themethod of claim 1, wherein prior to or as part of said detection in step(b) and/or step (c), each DNA region comprising said DMRs and/or saidother region(s), respectively, is(are) amplified.
 3. The method of claim1, wherein each detectable label used in step (b) and/or step (c) isindependently selected from the group consisting of: fluorescent,protein, small molecule or radioactive label.
 4. The method of claim 3,wherein each detectable label used in step (b) and step (c) isfluorescent.
 5. The method of claim 1, wherein two of said DMRs arelocated on different chromosomes.
 6. The method of claim 1, wherein saidother region is located between about 20 bp and about 20 kb upstream ordownstream of, and/or within the same gene as, at least one of saidDMRs.
 7. The method of claim 1, wherein said detection in step (c)comprises using at least two of said other regions; wherein, the numberof said other regions is the same as the number of DMRs used in step(b); and wherein, one of said other regions is located between about 20bp and about 20 kb upstream or downstream of a DMR used in step (b) andeach other of the said other regions is located between about 20 bp andabout 20 kb upstream or downstream of another of said DMRs.
 8. Themethod of claim 7, wherein: (A) said detection in step (c) is made usingthe same detectable label(s) for each of said other regions; and (B)said detection in step (c) comprises multiplex real-time quantitativeprobe-based PCR using at least two labelled probes each of which isspecific for one of said other regions.
 9. The method of claim 1,wherein said detection in step (c) and said detection in step (b) aremade using the same aliquot of DNA of said sample, and in the samereaction/detection vessel, and effectively simultaneously with eachother, and by multiplex real-time quantitative probe-based PCR using atleast one labelled probe specific for each of the said DMRs and otherregion(s).
 10. The method of claim 1, wherein said species of DNA iscirculating cell-free DNA and said sample is a plasma or serum sample.11. The method of claim 1, wherein said other region is located in aportion of the genome and/or gene selected from the group consisting of:GAPDH, beta-actin, ALB, APOE, RNASEP, RASSF1A, TBX3, HLCS, ZFY,CDC42EP1, MGC15523, SOX14 and SPN; and/or wherein, said other regioncomprises a region without a methylation site specific for said reagentand said locus is located in the genes RASSF1A or TBX3, and/or wherein,two or more of said other regions are used in detection step (c) andcomprise those located between about positions 14,220 bp and 13,350 bpof RASSF1A and about positions 12,400 bp and 13,000 bp of TBX3.
 12. Themethod of claim 1, wherein said other region is located between aboutpositions 12,400 bp and 13,000 bp of TBX3.
 13. The method of claim 1,wherein said pregnant female is susceptible to a pregnancy-associatedmedical condition selected from the group consisting of: preeclampsia,preterm labor, intrauterine growth retardation and vanishing twin. 14.The method of claim 13, wherein said pregnancy-associated medicalcondition is preeclampsia.
 15. The method of claim 1, wherein saidsample is a tissue sample or a sample of biological fluid selected fromthe group consisting of: whole blood, a blood fraction, urine, saliva,sweat, tears, phlegm, vaginal secretion, vaginal wash and colonic wash.16. The method of claim 15, wherein the sample is a plasma or serumsample.
 17. The method of claim 1, wherein said reagent thatdifferentially modifies methylated and non-methylated DNA comprisesbisulphite.
 18. The method of claim 1, wherein said reagent thatdifferentially modifies methylated and non-methylated DNA comprises anagent that selectively digests unmethylated over methylated DNA,wherein, said agent comprises: at least one methylation sensitiveenzyme; at least one methylation sensitive restriction enzyme; and/or anagent selected from the group consisting of: AatII, AciI, AclI, AfeI,AgeI, AgeI-HF, AscI, AsiSI, AvaI, BceAI, BmgBI, BsaAI, BsaHI, BsiEI,BsiWI, BsmBI, BspDI, BsrFI, BssHII, BstBI, BstUI, ClaI, EagI, FauI,FseI, FspI, HaeII, HgaI, HhaI, HinPlI, HpaII, Hpy99I, HpyCH4IV, KasI,MluI, NaeI, NarI, NgoMIV, NotI, NotI-HF, NruI, Nt.BsmAI, Nt.CviPII,PaeR7I, PluTI, PmlI, PvuI, PvuI-HF, RsrII, SacII, SalI, SalI-HF, SfoI,SgrAI, SmaI, SnaBI, TspMI and ZraI.
 19. The method of claim 18, whereinsaid reagent comprises at least one methylation sensitive restrictionenzyme and wherein: (i) said DMRs comprise at least one, or at leasttwo, site(s) of methylation recognized by said methylation sensitiverestriction enzyme; (ii) said other region does not comprise a site ofmethylation recognized by said methylation sensitive restriction enzyme.20. The method of claim 18, wherein said reagent comprises two or threeof the methylation sensitive restriction enzymes selected from the groupconsisting of BstUI, HhaI and HpaII.
 21. The method of claim 1, whereineach of said detection steps comprises quantitative detection and saiddetected amount of said species of DNA is expressed as a relativeconcentration of said species of DNA to the total DNA in said sample.22. The method of claim 21, further comprising the step: comparing theamount of said species of DNA detected with a threshold amount and/orreference distribution of amounts, wherein: (x) an increase in, oroutlying of, the amount of said species of DNA indicates an increasedrisk of the individual suffering from or developing a medical condition;and/or (y) an amount of said species of DNA in excess to said threshold,or outlying from said distribution, indicates that a diagnosis for anabnormality in the said species of DNA present in said sample may beperformed on said sample.
 23. The method of claim 21, further comprisingthe step: performing on said sample, a diagnosis for an abnormality insaid species of DNA present in said sample, wherein said diagnosis is aprenatal diagnosis.
 24. The method of claim 23, wherein said diagnosiscomprises a step that uses a detection technology selected from thegroup consisting of: DNA sequencing, SNP analysis, digital PCR andhybridization.
 25. The method of claim 23, wherein: said abnormality isa genetic mutation or a chromosomal abnormality associated with a fetalabnormality and/or a congenital disorder; wherein: said genetic mutationis selected from the group consisting of: color blindness, cysticfibrosis, hemochromatosis, hemophilia, phenylketonuria, polycystickidney disease, sickle-cell and disease, Tay-Sachs disease; and/or saidchromosomal abnormality is selected from the group consisting of: atrisomy, trisomy 21, trisomy 18, trisomy 13, a sex-chromosomeabnormality, Turners syndrome, Klinefelter syndrome, Noonan syndrome,Triple X syndrome, XXY syndrome, Fragile X syndrome, XYY syndrome, XXYYsyndrome, a chromosomal deletion, Prader-Willi syndrome, Cris-du-chatsyndrome, Wolf-Hirschhorn syndrome, 22q11 deletion syndrome, Duchenemuscular dystrophy, Beckwith-Wiedemann syndrome, Canvan syndrome, andneurofibromatosis.
 26. The method of claim 23, wherein said abnormalityis a chromosomal trisomy.
 27. The method of claim 26, wherein saidchromosomal trisomy is trisomy 21, trisomy 18, or trisomy
 13. 28. Amethod for detecting an increased risk of an individual suffering fromor developing a medical condition; said method comprising the steps: (i)conducting the method of claim 21; and (ii) comparing the amount of saidspecies of DNA detected with a threshold amount and/or a referencedistribution of amounts, wherein an increase in, or outlying of, theamount of said species of DNA indicates an increased risk of theindividual suffering from or developing said medical condition.
 29. Themethod of claim 28, wherein the individual is a pregnant femalesusceptible to or considered at risk of being susceptible to sufferingor developing a pregnancy-associated medical condition selected from thegroup consisting of preeclampsia, preterm labor, intrauterine growthretardation and vanishing twin.
 30. The method of claim 29, wherein saidpregnancy-associated medical condition is preeclampsia.
 31. The methodof claim, 21, further comprising the step: comparing the amount of saidspecies of DNA detected with a threshold amount and/or referencedistribution of amounts, wherein: (x) an increase in, or outlying of,the amount of said species of DNA indicates an increased risk of theindividual suffering from or developing a medical condition, wherein themedical condition is a pregnancy-associated medical condition selectedfrom the group consisting of: preeclampsia, preterm labor, intrauterinegrowth retardation and vanishing twin.
 32. The method of claim 31,wherein said pregnancy-associated medical condition is preeclampsia. 33.The method of claim 1, further comprising the steps: detecting an amountof total DNA in a standard sample of DNA of known amount using the sameother regions(s) as used in step (c); and comparing the signal detectedfrom said standard sample of DNA to the signal detected in step (c). 34.The method of claim 33, wherein each of said detection steps comprisesquantitative detection and said detected amount of said species of DNAis expressed as an absolute amount of said species of DNA in saidsample.
 35. The method of claim 1, wherein said DMRs comprise at leastone, or at least two, methylation site(s) specific for said reagent, andat least one of said DMRs is located in a region and/or gene selectedfrom the group consisting of SEQ ID NOs 15-187.
 36. The method of claim35, wherein at least one of said DMRs is located in a region and/or geneselected from the group consisting of: SEQ ID NOs 15-53 and 148-187. 37.The method of claim 1, wherein at least one of said DMRs is located onhuman chromosome
 12. 38. The method of claim 1, wherein said DMRscomprise at least one, or at least two, methylation site(s) specific forsaid reagent, and at least one of said DMRs is located in a regionand/or gene selected from the group consisting of: SEQ ID NOs 188-199.39. The method of claim 1, wherein at least one of said DMRs: is locatedon a human chromosome selected from the list consisting of: chromosome21, chromosome 18, and chromosome
 13. 40. The method of claim 1, whereinat least one of said DMRs is located in a region and/or gene selectedfrom the group consisting of: SEQ ID NOs 54-147.
 41. The method of claim40, wherein at least one of said DMRs is located in a region and/or geneselected from the group consisting of: SEQ ID NOs: 56, 66, 73, 87, 102,126 and
 143. 42. The method of claim 1, wherein at least one of saidDMRs: is located on a human chromosome selected from the groupconsisting of: chromosome 1 to 12, chromosome 14 to 17, chromosome 19,chromosome 20, chromosome 22 and chromosome
 23. 43. The method of claim42, wherein at least one of said DMRs is located on human chromosome 3or human chromosome
 19. 44. The method of claim 42, wherein at least oneof said DMRs is located on human chromosome
 5. 45. The method of claim1, wherein each of said DMRs is located in a region and/or geneindependently selected from the group consisting of: SEQ ID NOs 54-147.46. The method of claim 45, wherein at least one, or each, of said DMRsis located in a region and/or gene independently selected from the groupconsisting of: SEQ ID NOs 66, 102, and
 126. 47. The method of claim 1,wherein the other region is located upstream or downstream of one ofsaid DMRs within a distance selected from the group consisting of:between about 15 kb to 10 kb, 12 kb to 8 kb, 10 kb to 8 kb, 11 kb to 7kb, 11 kb to 10 kb, 9 kb to 8 kb, 8 kb to 6 kb, 6 kb to 4 kb, 4 kb to 2kb, and 2 kb to 500 bp.
 48. The method of claim 1, wherein two speciesof DNA are detected in said sample.
 49. The method of claim 48, wherein:in at least one detection step (b): the presence of methylated DNA at afirst set of two or more DMRs is used to indicate the presence of anamount of a first species of DNA in said sample and the absence ofmethylated DNA at said first set of DMRs indicates the absence of saidfirst species of DNA in said sample; wherein at least one of said firstset of DMRs is located in a region and/or gene selected from the groupconsisting of: SEQ ID NOs 15-53 and 148-187, or located in a regionand/or gene selected from the group consisting of: SEQ ID NOs 188-199;the presence of methylated DNA at a second set of two or more DMRs isused to indicate the presence of an amount of a second species of DNA insaid sample and the absence of methylated DNA at said second set of DMRsindicates the absence of said second species of DNA in said sample;wherein at least one of said second set of DMRs is located in a regionand/or gene selected from the group consisting of: SEQ ID NOs 54-147;and in at least one detection step (c): a first amount of total DNApresent in said sample is detected using a first region that is notdifferently methylated between said first species of DNA and the DNA notoriginating from cells of said type, the modification of which firstother region by said reagent is insensitive to methylation of DNA,wherein said first other region is located between about 20 bp and about20 kb upstream or downstream of at least one of said first set of DMRs;and a second amount of total DNA present in said sample is detectedusing a second region that is not differently methylated between saidsecond species of DNA and the DNA not originating from cells of saidtype, the modification of which second other region by said reagent isinsensitive to methylation of DNA, wherein said second other region islocated between about 20 bp and about 20 kb upstream or downstream of atleast one of said second set of DMRs.
 50. The method of claim 1, whereinat least one of said DMRs is located in a region and/or gene selectedfrom the group consisting of CD48, FAIM3, ARHGAP25, SELPLG, APC, CASP8,RARB, SCGB3A1, DAB2IP, PTPN6, THY1, TMEFF2 and PYCARD.
 51. The method ofclaim 1, wherein at least one of said DMRs is located in a region and/orgene selected from the group consisting of RASSF1A, TBX3, HLCS, ZFY,CDC42EP1, MGC15523, SOX14 and SPN.
 52. The method of claim 1, wherein atleast one of said DMRs is located in a portion of the genome and/or genethat is RASSF1A or TBX3.
 53. The method of claim 1, wherein at least oneof said DMRs is located between about positions 4,700 bp and 5,600 bp ofRASSF1A.
 54. The method of claim 53, wherein at least one of said DMRsis located between about positions 5,100 bp and 5,300 bp of RASSF1A. 55.The method of claim 1, wherein at least one of said DMRs is locatedbetween about positions 1,600 bp and 2,400 bp of TBX3.
 56. The method ofclaim 55, wherein at least one of said DMRs is located between aboutpositions 1,800 bp and 2,260 bp of TBX3.
 57. A method for detecting achromosomal aneuploidy in a fetus carried by a pregnant female, saidmethod comprising the steps: (A) determining, using a first method, in asample taken from said pregnant female the amount of a first species ofDNA that originates from cells of a fetus and/or the placenta of afetus, wherein said first species of DNA is located on a chromosomerelevant to the chromosomal aneuploidy or within a section of achromosome relevant to the chromosomal aneuploidy, and wherein saidfirst species of DNA that originates from cells of a fetus and/or theplacenta of a fetus is distinguished from its counterpart of maternalorigin in the sample due to differential DNA methylation; wherein saidfirst method is for quantitatively detecting in a sample from anindividual an amount of a species of DNA originating from cells of agiven type, which sample comprises said species of DNA in admixture withdifferently methylated DNA not originating from cells of said type,wherein said species of DNA originates from cells of a fetus and/or theplacenta of a fetus and said sample is from a pregnant female; saidfirst method comprising the steps: (a) treating the DNA present in saidsample with a reagent that differentially modifies methylated andnon-methylated DNA; (b) quantitatively detecting in said sample thepresence of methylation in said species of DNA at two or moredifferentially methylated regions (DMRs) that are differently methylatedbetween said species of DNA and the DNA not originating from cells ofsaid type, the modification of DNA of such DMRs by said reagent issensitive to methylation of DNA, wherein the presence of methylated DNAat one or more of said DMRs indicates the presence of said amount ofspecies of DNA in said sample and the absence of methylated DNA at saidDMRs indicates the absence of said species of DNA in said sample,wherein said DMRs are hyper-methylated in fetal DNA and hypo-methylatedin maternal DNA, and wherein said DMRs comprise at least one, or atleast two, methylation site(s) specific for said reagent, and at leastone of said DMRs is located in a region and/or gene selected from thegroup consisting of: SEQ ID NOs 15-53 and 148-187, or located in aregion and/or gene selected from the group consisting of: SEQ ID NOs188-199; (c) quantitatively detecting an amount of total DNA present insaid sample using at least one other region, wherein there is nodetectable difference between modification by said reagent at the otherregion(s) of DNA originating from cells of a fetus and/or the placentaof a fetus as compared to the other region(s) of maternal DNA, wherein,said detection in step (b) and said detection in step (c) are made usingthe same aliquot of DNA of said sample, and in the samereaction/detection vessel, and effectively simultaneously for such DMRsand other region(s), and using the same detectable labels(s) for each ofsaid DMRs and a different detectable label(s) for said other region(s)(B) determining, using the method of claim 1, the amount of a secondspecies of DNA that originates from cells of a fetus and/or the placentaof a fetus in said sample, wherein said second species of DNA is locatedon a reference chromosome, and wherein said second species of DNA thatoriginates from cells of a fetus and/or the placenta of a fetus isdistinguished from its counterpart of maternal origin in the sample dueto differential DNA methylation; (C) determining the relative amount, orthe ratio, of the amounts from (A) and (B); and (D) comparing saidrelative amount or ratio with a threshold and/or reference distributionof amount(s) or ratio(s), wherein: a relative amount or ratio that ishigher or lower than said threshold and/or reference distribution ofamount(s) or ratio(s) indicates the presence of the chromosomalaneuploidy in the fetus.